Use of ak6 in the preparation of a drug for treating uremic cardiomyopathy

Abstract

The application relates to application of an AK6 gene, AK6 protein or AK6 overexpression construct in preparation of a uremic cardiomyopathy treatment drug and belongs to the technical field of biological medicines. In order to solve the problem that treatment of uremic cardiomyopathy lacks effective and precise intervention targets, the application provides application of an AK6 gene, AK6 protein or AK6 overexpression construct in preparation of a uremic cardiomyopathy treatment drug, the nucleotide sequence of the AK6 gene is shown in SEQ ID NO:1, and the amino acid sequence of the AK6 protein is shown in SEQ ID NO:2. Through AAV9-mediated AK6 overexpression, it is confirmed that AK6 can be used as a precise target of uremic cardiomyopathy, overexpression of AK6 can improve heart function, reduce left ventricular wall thickening, inhibit myocardial cell hypertrophy and myocardial fibrosis, and can reduce mitochondrial ultrastructure damage, restore oxidative phosphorylation protein expression, improve mitochondrial respiratory function and reduce the level of mitochondrial active oxygen, and realize targeted protection of uremic cardiomyopathy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a gene therapy drug for treating uremic cardiomyopathy, and particularly to the application of the AK6 gene, AK6 protein, or AK6 overexpression construct in the preparation of a drug for treating uremic cardiomyopathy. Background Technology

[0002] Chronic kidney disease (CKD) is a major public health problem worldwide, with its prevalence continuously rising. It has become a significant risk factor for cardiovascular disease morbidity and mortality. Epidemiological studies show that the risk of cardiovascular death in CKD patients is significantly higher than in the general population, and cardiovascular complications are one of the leading causes of death in CKD patients. As an important manifestation of the interaction between the heart and kidneys, the structural and functional damage to the heart secondary to CKD typically manifests as left ventricular hypertrophy, myocardial fibrosis, impaired myocardial energy metabolism, mitochondrial dysfunction, and progressive decline in cardiac function. Clinically, this phenotype is defined as uremic cardiomyopathy. The pathogenesis of uremic cardiomyopathy is extremely complex, involving the interaction of multiple factors, including the accumulation of uremic toxins, a persistent inflammatory state, increased oxidative stress, impaired mitochondrial structure and function, and metabolic reprogramming of cardiomyocytes, ultimately leading to cardiomyocyte damage, cardiac remodeling, and heart failure.

[0003] Currently, clinical treatment strategies for uremic cardiomyopathy mainly include blood pressure control, anemia correction, volume overload management, mineral metabolism regulation, and supportive measures such as renal replacement therapy (hemodialysis, peritoneal dialysis, or kidney transplantation). These methods are all indirect interventions, primarily aimed at alleviating systemic pathophysiological disturbances in the uremic state, rather than directly addressing the mechanisms of cardiomyocyte damage. While existing treatments can slow disease progression to some extent, they are insufficient to effectively reverse existing myocardial damage and cardiac remodeling. More importantly, there is currently a lack of targeted interventions that can directly protect cardiomyocytes, improve mitochondrial function, and reduce cardiac remodeling. Therefore, identifying key pathogenic molecular targets for uremic cardiomyopathy and developing intervention strategies that can be precisely delivered to myocardial tissue has significant scientific and clinical translational value.

[0004] In recent years, gene therapy has made significant progress in the field of cardiovascular diseases. Adeno-associated virus (AAV) has become the most promising delivery vector for gene therapy in cardiovascular diseases due to its advantages such as good safety, low immunogenicity, and ability to achieve long-term gene expression in non-dividing cells. Among them, the AAV9 serotype can achieve efficient systemic delivery through intravenous injection and can preferentially transduce myocardial tissue. To reduce the risk of off-target expression in non-target organs such as the liver, myocardial-specific promoters (such as the cardiac troponin T promoter, cTnT) are usually used to drive the selective expression of therapeutic genes in cardiomyocytes. Based on the above technical strategies, researchers have developed AAV9-mediated gene therapy regimens for different types of heart diseases. For example, some studies have used AAV9-cTnT-Aralar to treat heart failure by improving mitochondrial function, some studies have used AAV9-cTnT-ACLY to treat cardiomyopathy by regulating protein acetylation, and some studies have used AAV9-cTnT-OGN to prevent myocardial ischemia-reperfusion injury by regulating mitochondrial dynamics. All of the above schemes use the AAV9-cTnT vector system, but the target genes are all molecules known to be related to cardiac function or mitochondrial metabolism.

[0005] AK6 (adenylate kinase 6, also known as hCINAP) is a member of the atypical adenosine kinase family, structurally different from typical adenosine kinases. Current research indicates that AK6 primarily participates in the regulation of cellular energy and nucleotide metabolism, involving the phosphate transfer reaction between ATP and AMP. Current understanding of AK6 function mainly focuses on its role in tumor cell proliferation regulation, early embryonic development, and the regulation of inflammatory signaling pathways. Studies have reported aberrant expression of AK6 in various tumors, including lung cancer and colorectal cancer, and its involvement in tumor cell energy metabolism reprogramming. However, to date, no studies have reported an association between AK6 and cardiac diseases (especially uremic cardiomyopathy), nor have any studies suggested that AK6 could serve as a target for cardioprotective intervention. Regarding the role of AK6 in the regulation of mitochondrial function in cardiomyocytes, and whether its overexpression can improve myocardial damage under uremic stress, there is a complete lack of relevant documentation in the current technology.

[0006] In summary, the existing technologies have the following main shortcomings: First, the treatment of uremic cardiomyopathy lacks effective interventions that can directly target mitochondrial damage and metabolic disorders in cardiomyocytes. Existing treatment plans are all indirect supportive measures, which are difficult to reverse or prevent the progression of myocardial damage. Second, although the AAV9-cTnT vector system has been applied in the field of cardiac gene therapy, the molecules it targets are all known cardiac function-related genes. Third, the value of AK6 as a therapeutic target for uremic cardiomyopathy, and the feasibility of treating uremic cardiomyopathy by AK6 myocardial overexpression mediated by AAV9, have not been mentioned or suggested in the existing technologies. Summary of the Invention

[0007] To address the shortcomings of existing treatments for uremic cardiomyopathy, such as the lack of effective and precise intervention targets and the fact that current treatments are mostly indirect supportive measures and cannot directly protect the mitochondrial function of cardiomyocytes, this invention provides the application of the AK6 gene, AK6 protein, or AK6 overexpression constructs in the preparation of therapeutic drugs for uremic cardiomyopathy. This invention, through an adeno-associated virus 9 (AAV9)-mediated AK6 myocardial-specific overexpression system, is the first to demonstrate the core value of AK6 as a precise target for uremic cardiomyopathy.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the application of the AK6 gene, AK6 protein, or AK6 overexpression construct in the preparation of a therapeutic drug for uremic cardiomyopathy.

[0009] Furthermore, the nucleotide sequence of the AK6 gene is shown in SEQ ID NO:1, and the amino acid sequence of the AK6 protein is shown in SEQ ID NO:2.

[0010] Furthermore, the AK6 overexpression construct is a recombinant adeno-associated virus vector. Adeno-associated viruses have advantages such as good safety, low immunogenicity, and long-term stable expression in cardiomyocytes, making them suitable as gene delivery vectors.

[0011] Furthermore, the serotype of the recombinant adeno-associated virus vector is AAV9. The AAV9 serotype, after intravenous injection, can efficiently transduce myocardial tissue and exhibits excellent cardiac-targeted delivery characteristics.

[0012] Furthermore, the recombinant adeno-associated virus vector contains a myocardium-specific promoter. Using a myocardium-specific promoter allows the AK6 gene to be expressed only in cardiomyocytes, avoiding the risk of off-target expression in non-target organs such as the liver.

[0013] Furthermore, the myocardial-specific promoter is the cardiac troponin T promoter. This promoter exhibits highly specific transcriptional activation activity in cardiomyocytes.

[0014] Furthermore, the AK6 overexpression vector is an AAV9-cTnT-AK6 expression vector, which contains a nucleotide sequence encoding AK6 (SEQ ID NO:1) and a cardiac troponin T promoter.

[0015] Furthermore, the drug for treating uremic cardiomyopathy has at least one of the following uses: (1) improving cardiac function in uremic cardiomyopathy; (2) reducing left ventricular wall thickening, inhibiting myocardial hypertrophy and myocardial fibrosis; (3) reducing mitochondrial ultrastructural damage, restoring the expression of oxidative phosphorylation-related proteins, improving mitochondrial respiratory function and reducing mitochondrial reactive oxygen species levels.

[0016] Specifically, the improvement of cardiac function in uremic cardiomyopathy includes increasing ejection fraction and fractional shortening. The restoration of oxidative phosphorylation-related protein expression includes restoring the expression levels of at least one of the following proteins: ATP5A1, UQCRC2, SDHB, MTCO2, and NDUFB8. The improvement of mitochondrial respiratory function includes increasing basal oxygen consumption and / or maximal oxygen consumption.

[0017] The above effects have been verified in a 5 / 6 nephrectomy mouse model: After tail vein injection of AAV9-cTnT-AK6, the ejection fraction and short axis shortening rate of mice were significantly increased, and the thickness of the left ventricular wall was reduced; myocardial fibrosis, cardiomyocyte hypertrophy and tissue structural damage were significantly reduced; at the same time, ultrastructural damage such as mitochondrial swelling and cristae disorder was significantly improved, the expression of oxidative phosphorylation-related proteins such as ATP5A1, UQCRC2, SDHB, MTCO2 and NDUFB8 was restored, mitochondrial oxygen consumption rate was increased and reactive oxygen species level was reduced.

[0018] Furthermore, the uremic cardiomyopathy is myocardial damage secondary to chronic kidney disease, involving mitochondrial structural and functional disorders caused by the accumulation of uremic toxins.

[0019] In a second aspect, the present invention also provides a pharmaceutical composition for treating uremic cardiomyopathy, comprising the above-mentioned AK6 gene, AK6 protein or AK6 overexpression construct, and at least one of a pharmaceutically acceptable carrier, diluent, buffer system or stabilizer.

[0020] Furthermore, the AK6 overexpression construct is a recombinant adeno-associated virus vector AAV9-AK6, containing a nucleotide sequence encoding AK6 and a myocardial-specific promoter. The nucleotide sequence encoding AK6 is shown in SEQ ID NO:1, and the myocardial-specific promoter is the cardiac troponin T promoter.

[0021] Furthermore, the pharmaceutical composition is in the form of an injection, administered via intravenous injection. Intravenous injection enables efficient delivery of the AAV9 carrier to the heart.

[0022] Compared with the prior art, the present invention has the following outstanding advantages: (1) This invention is the first to demonstrate that AK6 can serve as a precise therapeutic target for uremic cardiomyopathy. In the prior art, AK6 is only involved in the fields of tumors, early embryonic development and inflammatory response, and its functional research in heart diseases is still blank. This invention is the first to discover that AK6 is significantly downregulated in uremic cardiomyopathy and achieves targeted protection against uremic cardiomyopathy through AAV9-mediated myocardial overexpression.

[0023] (2) This invention provides a complete chain of positive and negative genetic evidence for “downregulation-overexpression protection-knockout aggravation”. Not only has it been demonstrated in a 5 / 6 nephrectomy mouse model that AK6 overexpression can improve cardiac function, reduce pathological damage and mitochondrial dysfunction, but it has also been further verified in a cardiomyocyte-specific AK6 knockout mouse model that AK6 deficiency aggravates the phenotype of uremic cardiomyopathy, providing a solid genetic basis for AK6 as a therapeutic target.

[0024] (3) This invention reveals for the first time that AK6 overexpression exerts cardioprotective effects through a three-dimensional synergistic mechanism—simultaneously restoring the expression of oxidative phosphorylation-related proteins (ATP5A1, UQCRC2, SDHB, MTCO2, NDUFB8), improving mitochondrial respiratory function, and reducing mitochondrial reactive oxygen species levels. This combination of mechanisms has not been disclosed or suggested in the prior art.

[0025] (4) The present invention uses the AAV9-cTnT vector system to achieve myocardial-specific delivery and expression of the AK6 gene, which not only ensures the effective enrichment of the therapeutic gene in the target organ myocardium, but also reduces the off-target risk in non-target organs, and has good safety and clinical translation potential.

[0026] (5) This invention fills a gap in the field of gene therapy for uremic cardiomyopathy. Existing treatments for uremic cardiomyopathy are all indirect supportive measures such as controlling blood pressure, correcting anemia, and renal replacement therapy, lacking interventions that directly target mitochondrial damage in cardiomyocytes. This invention provides a novel gene therapy strategy for the treatment of uremic cardiomyopathy with AK6 as a precise target.

[0027] In summary, this invention provides a targeted therapy for uremic cardiomyopathy based on the AK6 gene, AK6 protein, or AK6 overexpression construct, which has a clear target basis, complete experimental evidence support, and good prospects for clinical translation. Attached Figure Description

[0028] Figure 1 This image shows the expression level of AK6 in a 5 / 6 nephrectomy mouse model. Figure 1A shows echocardiographic images of the heart and AK6 immunohistochemical staining of myocardial tissue in mice in the sham surgery group (Sham) and the 5 / 6 nephrectomy group (5 / 6Nx). Figure 1 B is a Western blot image of AK6 protein in myocardial tissue; Figure 1 C is a quantitative statistical graph of the relative expression level of AK6 protein; Figure 1 D is a quantitative statistical graph of the relative expression level of AK6 mRNA; Figure 2 The diagram shows the construction of an AAV9-mediated AK6 overexpression vector and its application in cardiac function testing. Figure 2 A shows the plasmid configuration of the recombinant AAV9-cTnT-AK6 vector; Figure 2 B is a fluorescence microscopy image of cells infected with AAV; Figure 2 C is a Western blot image showing AK6 protein expression after AAV infection; Figure 2 D represents representative echocardiographic images of mice in each group; Figure 2 E is a quantitative statistical chart of ejection fraction (EF%); Figure 2 F is a quantitative statistical chart of the minor axis shortening rate (FS%); Figure 2 G is a quantitative statistical graph of the diastolic thickness of the left ventricular posterior wall (LVPW d); Figure 2 H is a quantitative statistical graph of the systolic thickness of the left ventricular posterior wall (LVPW s); Figure 3 This is a histopathological image showing the effects of AAV9-mediated AK6 overexpression. Figure 3 A shows histological images of the heart of each group of mice after gross HE staining, as well as local HE staining, Masson staining, and WGA staining. Figure 3 B is a quantitative statistical graph of the cross-sectional area (CSV) of cardiomyocytes; Figure 3 C is a quantitative statistical graph of the proportion of myocardial fibrosis area; Figure 4 This image shows the gene construction and cardiac function testing of cardiomyocyte-specific AK6 knockout (AK6-cKO) mice. Figure 4 A is a schematic diagram of the gene knockout strategy for AK6-cKO mice; Figure 4 B is a Western blot diagram showing the expression of AK6 protein in the myocardium of wild-type (WT) and AK6-cKO mice; Figure 4 C represents representative echocardiographic images of mice in each group; Figure 4 D is a quantitative statistical chart of ejection fraction (EF%); Figure 4 E is a quantitative statistical chart of minor axis shortening rate (FS%); Figure 4 F is a quantitative statistical graph of the systolic thickness of the left ventricular posterior wall (LVPW s); Figure 4G is a quantitative statistical graph of the diastolic thickness of the left ventricular posterior wall (LVPW d); Figure 5 This image shows the histopathological examination of cardiomyocytes after specific AK6 knockout. Figure 5 A shows histological images of the hearts of mice in each group stained with HE, Masson, and WGA. Figure 5 B is a quantitative statistical chart showing the proportion of myocardial fibrosis area; Figure 5 C is a quantitative statistical graph of the cross-sectional area of ​​cardiomyocytes (CSV); Figure 6 This is a graph illustrating the effect of AK6 silencing on gene expression in cardiomyocytes. Among them, Figure 6 A is a volcano diagram of differentially expressed genes after AK6 silencing; Figure 6 B is a bubble chart showing the enrichment analysis of the KEGG signaling pathway for differentially expressed genes; Figure 7 This image shows the effects of AK6 overexpression on the structure and function of myocardial mitochondria under uremic stress. Among them, Figure 7 A shows transmission electron microscopy (TEM) images of myocardial mitochondria in each group of mice; Figure 7 B is a heatmap of gene expression related to mitochondrial oxidative phosphorylation; Figure 7 C is a Western blot image of proteins related to oxidative phosphorylation (OXPHOS); Figure 7 D is a quantitative statistical graph of the relative expression levels of OXPHOS-related proteins; Figure 8 This image shows the effects of cardiomyocyte-specific AK6 knockout on mitochondrial structure and function under uremic stress. Figure 8 A shows transmission electron microscopy (TEM) images of myocardial mitochondria in each group of mice; Figure 8 B is a heatmap of gene expression related to mitochondrial oxidative phosphorylation; Figure 8 C is a Western blot image of proteins related to oxidative phosphorylation (OXPHOS); Figure 8 D is a quantitative statistical graph of the relative expression levels of OXPHOS-related proteins; Figure 9 This is a map of the plasmid for the recombinant AAV9-cTnT-AK6 expression vector. Figure 9 A is the empty vector map (before the insertion of the target gene), showing the element structure of the backbone plasmid GL3058 pcAAV-cTNTo-GdGreen-WPRE, which includes the cardiac troponin T promoter (cTNTo), the green fluorescent protein reporter gene (GdGreen), and the WPRE post-transcriptional regulatory element; Figure 9B is the full map of the recombinant AAV9-cTnT-AK6 expression vector, showing the complete plasmid structure after inserting the AK6 coding sequence (SEQ ID NO:1) downstream of the cTNTo promoter. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to the embodiments. It should be understood that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Without departing from the concept of the present invention, those skilled in the art can routinely adjust the relevant experimental conditions, vector construction methods, drug dosages, and detection indicators. In the following embodiments, experimental data are expressed as mean ± SEM. Comparisons between two groups were performed using a two-tailed Student's t-test, and comparisons among multiple groups were performed using one-way ANOVA followed by Tukey's multiple comparison test. P < 0.05 was considered statistically significant.

[0030] The sequence is as follows: SEQ ID NO:1 (AK6 coding sequence, CDS, mouse, wild-type sequence): ATGAAGCTTCCGAATATCCTGCTCACCGGTACACCAGGGGTTGGAAAAACCACACTAGGCAAAGAACTTGCATCAAGATCAGGCCTGAAGTACGTTAACGTGGGAGATCTAGCTCGAGAAGGGCAGTTAT ACGACGGCTACGATGAGGAGTATGGCTGTCCCATTCTAGATGAGGATAGGGTTGTCGATGAGTTAGAGCATCAGATGCAGGAGGGCGGTGTGATTGTGGATTACCACGGCTGTGACTTCTTCCCCGAACG CTGGTTTCATAGTGTTTGTGTTGAGAACAGATAATGGTGTATTGTATAAACGACTTGAAACAAGGGGTTATAATGAGAAGAAACTACAAGACAATATTCAGTGTGAGATTTTTCAAGTTCTTTATGAA GAAGCCATAGCATCTTACAAAGAAGAAATCGTGCATCAGCTGCCCAGTAATGAACCAGAACAGCTCGAGGACAATATCAACCAAATCTCAAAATGGATTGAGCAGTGGGTCAAAGATCATAACCCTTAA.

[0031] SEQ ID NO:2 (AK6 protein amino acid sequence): MKLPNILLTGTPGVGKTTLGKELASRSGLKYVNVGDLAREGQLYDGYDEEYGCPILDEDRVVDELEHQMQEGGVIVDYHGCDFFPERWFHIVFVLRTDNGVLYKRLETRGYNEKKLQDNIQCEIFQVLYEEAIASYKEEIVHQLPSNEPEQLEDNINQISKWIEQWVKDHNP.

[0032] Animal experiments were approved by the Experimental Animal Management and Use Committee of Nanjing Medical University (ethics approval number: IACUC-2302033).

[0033] Example 1 Myocardial AK6 expression decreases under uremia stress.

[0034] A 5 / 6 nephrectomy mouse model was established. The experimental animals were divided into a sham-operated group and a 5 / 6 nephrectomy group, and heart tissue was collected 12 weeks post-operation. Immunohistochemistry, Western blot, and qPCR were used to detect the expression level of AK6 in myocardial tissue. The results showed that compared with the sham-operated group, the expression of both AK6 protein and mRNA in the myocardial tissue of the 5 / 6 nephrectomy group was significantly decreased (P<0.05), indicating that uremic stress is associated with decreased myocardial AK6 expression. The above results are as follows: Figure 1 As shown. Among them, Figure 1 Immunohistochemical staining of A showed that the AK6 positive signal in the myocardial tissue of the 5 / 6Nx group was weaker than that in the Sham group; Figure 1 Western blot bands of B showed that the AK6 protein expression level in the 5 / 6Nx group was lower than that in the Sham group; Figure 1 C and Figure 1 The quantitative statistical plot of D showed that the levels of AK6 protein and mRNA in the 5 / 6Nx group were lower than those in the Sham group.

[0035] Example 2 Construction of the recombinant AAV9-cTnT-AK6 expression vector.

[0036] A recombinant AAV9 expression vector carrying the AK6 coding sequence was constructed. The AK6 coding sequence is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2. The AK6 coding sequence was cloned into the AAV backbone plasmid GL3058 pcAAV-cTNTo-GdGreen-WPRE via HindIII and KpnI restriction endonuclease sites, placing it downstream of the cardiomyocyte-specific cTnT promoter (cTNTo, derived from mice, its sequence is described in the AAV-Heart System Technical Manual by Heyuan Biotechnology, this promoter is a known sequence in the art), thereby forming the AAV9-cTnT-AK6 expression vector. The empty control vector is designated NC-AAV9. The AAV9-cTnT-AK6 expression vector, after packaging, can be used for in vivo administration to enhance AK6 expression in cardiomyocytes.

[0037] The recombinant adeno-associated virus AAV9-cTnT-AK6 described in this embodiment was constructed and packaged by Heyuan Biotechnology Co., Ltd. based on its AAV-Heart system, and its construction name is pcAAV-cTNTo-HA-Ak6-P2A-Cre-WPRE. The PCR amplification primer sequences designed based on the AK6 gene sequence are as follows: the forward sequencing primer is 3058-F (SEQ ID NO:3), with the sequence CCTTTCTCTCCCACAGGTGTCCA; the reverse sequencing primer is WPRE-R (SEQ ID NO:4), with the sequence CATAGCGTAAAAGGAGCAACA.

[0038] The vector structure of the recombinant adeno-associated virus AAV9-cTnT-AK6 described in this embodiment is as follows: Figure 9 A (empty carrier map) and Figure 9 As shown in B (full map of the recombinant vector). The recombinant plasmid was sequenced using primers 3058-F and WPRE-R for verification. The sequencing results confirmed that the AK6 coding sequence was correctly inserted downstream of the cTNTo promoter, and the sequence was consistent with SEQ ID NO:1. Figure 2 Fluorescence microscopy images of B showed that obvious green fluorescence signals were visible in the cells of the AK6-AAV9 vector group, while no obvious fluorescence signals were seen in the NC-AAV9 control group; Figure 2 Western blot analysis of C showed that the expression of AK6 protein in the AK6-AAV9 vector group was significantly higher than that in the NC-AAV9 group, indicating that the AAV9-cTnT-AK6 vector can effectively mediate the expression of AK6 protein.

[0039] Example 3 AAV9-mediated myocardial overexpression of AK6 improves cardiac function in uremic cardiomyopathy.

[0040] Mice were randomly divided into four groups: sham surgery + control AAV group, sham surgery + AK6-AAV group, 5 / 6 nephrectomy + control AAV group, and 5 / 6 nephrectomy + AK6-AAV group. AAV9 vector was injected via tail vein at a volume of 100 μL and a dose of 1 × 10^11 vg. Echocardiography was performed 12 weeks post-surgery.

[0041] The results showed that compared with the 5 / 6 nephrectomy + control AAV group, the 5 / 6 nephrectomy + AK6-AAV group had significantly increased ejection fraction and fractional shortening, and decreased left ventricular posterior wall thickness, with statistically significant differences (P<0.05). This suggests that AAV9-mediated AK6 myocardial overexpression can improve uremic stress-induced cardiac systolic dysfunction and left ventricular wall thickening. The above results correspond to... Figure 2 .in, Figure 2 The ultrasound images of D show the changes in left ventricular cavity diameter and ventricular wall motion in each group; Figure 2 E to Figure 2 The quantitative statistical charts of H show the specific trends of EF%, FS%, LVPW d and LVPW s in each group. The data distribution shows that there are differences in cardiac function indicators between the 5 / 6Nx+AK6-AAV group and the 5 / 6Nx+NC-AAV group.

[0042] Example 4 AAV9-mediated myocardial overexpression of AK6 alleviates pathological changes in uremic cardiomyopathy.

[0043] HE staining, Masson staining, and WGA staining were performed on the heart tissues of the mice in each group described in Example 3. The results showed that the 5 / 6 nephrectomy + control AAV group exhibited significant myocardial structural disorder, increased collagen deposition, and increased cardiomyocyte cross-sectional area; in contrast, the 5 / 6 nephrectomy + AK6-AAV group showed significantly reduced pathological changes.

[0044] These results indicate that myocardial AK6 overexpression can alleviate myocardial fibrosis, cardiomyocyte hypertrophy, and tissue structural damage in uremic cardiomyopathy. The above results correspond to... Figure 3 . Figure 3 HE staining, Masson staining and WGA staining images of A show the morphology and fibrosis distribution of myocardial tissue in each group; Figure 3 B and Figure 3 The quantitative statistical graphs of C reflect the numerical changes in the cross-sectional area and fibrosis area of ​​cardiomyocytes in each group.

[0045] Example 5 Cardiac cell-specific AK6 knockout exacerbates cardiac dysfunction in uremic cardiomyopathy.

[0046] Cardiac cell-specific AK6 knockout mice are made by knocking out AK6 (flox / flox)Mouse and cardiomyocyte-specific Cre tool mouse (B6 / JGpt-H11) (em1Cin(Myh6-iCre) The AK6 knockout mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were hybridized to specifically express Cre recombinase in cardiomyocytes under the Myh6 promoter. Western blot analysis confirmed a significant decrease in myocardial AK6 protein. Wild-type mice and cardiomyocyte-specific AK6 knockout mice underwent sham surgery or 5 / 6 nephrectomy, respectively, and cardiac function was assessed 12 weeks post-surgery.

[0047] The results showed that, in the context of 5 / 6 nephrectomy, cardiomyocyte-specific AK6 knockout mice exhibited more significant decreases in ejection fraction, fractional shortening, and left ventricular wall thickening compared to the corresponding control mice (P<0.05), suggesting that AK6 deficiency exacerbates cardiac dysfunction in uremic cardiomyopathy. The above results correspond to... Figure 4 . Figure 4 Ultrasound images of C show changes in the heart chambers and ventricular walls of WT and AK6-cKO mice under 5 / 6Nx and Sham backgrounds; Figure 4 D to Figure 4 The quantitative statistical graph of G shows the data distribution characteristics of EF%, FS%, LVPW s, and LVPW d for each group.

[0048] Example 6 Cardiac cell-specific AK6 knockout exacerbates the pathological changes in uremic cardiomyopathy.

[0049] Histopathological analysis was performed on the cardiac tissues of the mice in each group described in Example 5. The results showed that, in the context of 5 / 6 nephrectomy, the cardiomyocyte-specific AK6 knockout mice exhibited more significant myocardial fibrosis, increased collagen deposition, and increased cardiomyocyte cross-sectional area compared with the corresponding control mice (P<0.05).

[0050] These results indicate that AK6 deficiency in cardiomyocytes promotes myocardial hypertrophy and myocardial fibrosis under uremic stress. The above results correspond to... Figure 5 .in, Figure 5 Histological images of A show the staining of tissue sections from WT and AK6-cKO mice against a 5 / 6Nx and Sham background; Figure 5 B and Figure 5 The quantitative statistical graphs of C compared the numerical differences in the area of ​​myocardial fibrosis and the cross-sectional area of ​​myocardial cells among the groups.

[0051] Example 7 AK6 silencing leads to metabolic reprogramming of cardiomyocytes.

[0052] Proteomic analysis was performed on neonatal rat ventricular myocytes transfected with control siRNA or si-AK6. The results showed that AK6 silencing led to the enrichment of differentially expressed proteins primarily in the tricarboxylic acid cycle, riboflavin metabolism, thiamine metabolism, and other metabolically related pathways, suggesting that AK6 deficiency can inhibit tricarboxylic acid cycle-related metabolic processes. These results correspond to… Figure 6 . Figure 6 The volcano plot of A shows the distribution of differentially expressed genes in cardiomyocytes after AK6 silencing; Figure 6 The KEGG bubble plot of B shows the biological pathways where differentially expressed genes are primarily enriched.

[0053] Example 8 AK6 overexpression protects myocardial mitochondrial structure and function under uremic stress.

[0054] Transmission electron microscopy was used to observe the ultrastructure of myocardial mitochondria. The results showed that 5 / 6 nephrectomy led to mitochondrial swelling, cristae disorder, and structural damage, while AK6 overexpression significantly alleviated the above-mentioned mitochondrial ultrastructural damage.

[0055] Western blot analysis showed that the expression of oxidative phosphorylation-related proteins such as ATP5A1, UQCRC2, SDHB, MTCO2, and NDUFB8 was decreased in the myocardium after 5 / 6 nephrectomy, while AK6 overexpression restored their expression levels (P<0.05). The above results correspond to... Figure 7 .in, Figure 7 Electron microscopy images of A show the ultrastructural changes of myocardial mitochondria in each group; Figure 7 The heatmap in B shows the expression clustering of genes related to mitochondrial oxidative phosphorylation in each group; Figure 7 Western blots and quantitative statistical plots for C and 7D respectively reflect the changes in the intensity and relative abundance of expression bands of ATP5A1, UQCRC2, SDHB, MTCO2, and NDUFB8 proteins in each group.

[0056] Example 9 AK6 knockout promotes mitochondrial structural and functional damage under uremic stress.

[0057] Under uremic stress, the ultrastructure and function of mitochondria in a cardiomyocyte-specific AK6 knockout model were evaluated. The results showed that AK6 deficiency further aggravated mitochondrial swelling, cristae breakage, and structural disorder, accompanied by a further decrease in oxidative phosphorylation-related proteins and an increase in mitochondrial reactive oxygen species levels (P<0.05).

[0058] This result further demonstrates that AK6 plays an important role in maintaining myocardial mitochondrial homeostasis under uremic stress. The above results correspond to... Figure 8 .in, Figure 8Electron microscopy images of A show the differences in mitochondrial structure between WT and AK6-cKO mice; Figure 8 The heatmap in B reflects the clustering comparison of expression levels of mitochondrial-related genes in each group; Figure 8 Western blots and quantitative statistical plots for C and 8D show the quantitative changes in expression bands and relative abundance of OXPHOS-related proteins in each group.

[0059] In summary, this invention is based on Figures 1 to 9 The experimental results demonstrate that AK6 expression is reduced in myocardial tissue and cardiomyocytes under uremic stress. AAV9-mediated AK6 myocardial overexpression can improve cardiac function and pathological damage in uremic cardiomyopathy and protect mitochondrial structure and function; cardiomyocyte-specific AK6 deficiency aggravates the uremic cardiomyopathy phenotype. Therefore, the AAV9-cTnT-AK6 expression vector and its drug composition can serve as candidate gene therapy for the prevention, alleviation, or treatment of uremic cardiomyopathy.

[0060] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Application of AK6 gene, AK6 protein or AK6 overexpression construct in the preparation of drugs for the treatment of uremic cardiomyopathy.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the AK6 gene is shown in SEQ ID NO:1, and the amino acid sequence of the AK6 protein is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, The AK6 overexpression construct is a recombinant adeno-associated virus vector.

4. The application according to claim 3, characterized in that, The serotype of the recombinant adeno-associated virus vector is AAV9.

5. The application according to claim 3, characterized in that, The recombinant adeno-associated virus vector contains a myocardial-specific promoter.

6. The application according to claim 5, characterized in that, The myocardial-specific promoter is the cardiac troponin T promoter.

7. The application according to any one of claims 1-6, characterized in that, The drug for treating uremic cardiomyopathy has at least one of the following uses: (1) Improves cardiac function in uremic cardiomyopathy; (2) Reduces left ventricular wall thickening and inhibits myocardial cell hypertrophy and myocardial fibrosis; (3) Reduce mitochondrial ultrastructural damage, restore the expression of oxidative phosphorylation-related proteins, improve mitochondrial respiratory function and reduce mitochondrial reactive oxygen species levels.

8. A pharmaceutical composition for treating uremic cardiomyopathy, characterized in that, It comprises the AK6 gene, AK6 protein or AK6 overexpression construct as described in any one of claims 1-7, and at least one of a pharmaceutically acceptable vector, diluent, buffer system or stabilizer.

9. The pharmaceutical composition according to claim 8, characterized in that, The AK6 overexpression construct is a recombinant adeno-associated virus vector AAV9-AK6, containing a nucleotide sequence encoding AK6 and a myocardial-specific promoter. The nucleotide sequence encoding AK6 is shown in SEQ ID NO:

1.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that, The pharmaceutical composition is in the form of an injection and is administered via intravenous injection.

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