Potential therapeutic target for cardiac hypertrophy and heart failure

Abstract

The invention provides a potential therapeutic target for cardiac hypertrophy and heart failure, finds that the cardiac hypertrophy caused by pressure load can be obviously relieved by using adeno-associated virus (AVV9) as a carrier myocardial cell specific overexpression Ces1d gene, and finds that the Ces1d relieves the cardiac hypertrophy by promoting fatty acid oxidation; and a potential target and a new strategy are provided for heart failure treatment. According to the invention, Ces1d is taken as a regulation target, and the expression level of Ces1d is regulated, so that the fatty acid oxidation of myocardial cells can be obviously promoted, and the expression of fatty acid oxidation related genes such as Cpt1a, Cpt1b, Cpt2, Acadm, Acadvl and Acox1 can be up-regulated, thereby optimizing the utilization mode of a myocardial energy metabolism substrate; meanwhile, mitochondrial dysfunction and mitochondrial generation damage induced by pressure load can be improved. Therefore, the regulation strategy taking the Ces1d as the action target and the AVV9 as the carrier provides a new gene therapy technical approach for prevention and treatment of cardiac hypertrophy and related cardiovascular diseases, and has a good application prospect and a remarkable clinical potential value.

Description

Technical Field

[0001] This invention belongs to the field of disease diagnosis and treatment biomarker screening and application technology, specifically relating to a potential therapeutic target for myocardial hypertrophy and heart failure. Background Technology

[0002] Rapid population aging is an increasingly serious social problem globally. my country, a populous country, has entered a moderately aging stage, and the incidence of diseases common to the elderly, such as cardiovascular disease (CVD), has reached unprecedented levels, and is accelerating and trending towards affecting younger people, becoming a serious public health and clinical issue. Currently, my country has 330 million CVD patients. Various types of CVD, including hypertension, coronary heart disease, cardiomyopathy, and valvular heart disease, can cause cardiac overload, leading to myocardial hypertrophy and ultimately chronic heart failure. Conservative estimates suggest that my country currently has 8.9 million chronic heart failure patients, with a large number of potential patients. Therefore, the prevention and treatment of chronic heart failure remains a challenging task, and the disease burden is increasing, making it a major public health problem in my country. Myocardial hypertrophy is an early stage of heart failure. Reducing myocardial hypertrophy can significantly lower the incidence of heart failure and delay its progression, providing possibilities for early treatment. Currently, there are no specific drugs for myocardial hypertrophy and heart failure; therefore, reducing myocardial hypertrophy and delaying the development of heart failure points the way to the treatment of heart failure. Based on this, conducting relevant research to explore the pathogenesis of myocardial hypertrophy caused by stress overload, and early prevention, identification and intervention of heart failure caused by myocardial hypertrophy are still urgent needs in my country's current aging society, and have important social value and scientific significance.

[0003] Metabolic remodeling accompanies myocardial hypertrophy. Under physiological conditions, adult cardiomyocytes primarily generate ATP through fatty acid oxidation, meeting approximately 95% of their energy needs, with 70% of this being long-chain fatty acids. In myocardial hypertrophy induced by long-term stress load, hypoxia, and other pathological conditions, the energy supply for cardiomyocytes gradually shifts from fatty acid oxidation to glucose and other substrate metabolism. In the late or terminal stages, fatty acid oxidation is significantly downregulated. Current research suggests that preserving fatty acid oxidation during long-term stress load can enhance myocardial contractile function and reduce myocardial hypertrophy, providing new ideas and strategies for the treatment of myocardial hypertrophy and heart failure. Studying the effects and mechanisms of myocardial metabolic remodeling and fatty acid oxidation on myocardial hypertrophy may ultimately achieve the goal of protecting and improving myocardial function, delaying or even treating heart failure. This is also a current research hotspot in the cardiovascular field. Summary of the Invention

[0004] This invention provides a potential therapeutic target for myocardial hypertrophy and heart failure. This invention discovers that cardiomyocyte-specific overexpression of the Ces1d gene can significantly reduce stress-induced myocardial hypertrophy. It also discovers that Ces1d reduces myocardial hypertrophy by promoting fatty acid oxidation, providing a potential target and new strategy for the treatment of heart failure.

[0005] This invention first provides one use of the Ces1d gene as a potential therapeutic target for myocardial hypertrophy and heart failure; The Ces1d gene described herein has the following specific amino acid sequence: (SEQ ID NO:1); Its encoded nucleotide sequence is as follows: The present invention also provides an article for the prevention or treatment of myocardial hypertrophy and heart failure, the article comprising an article capable of increasing the expression level of the Ces1d gene; The product described herein, as a specific example, is a gene recombination expression vector; The recombinant expression vector is a cardiomyocyte-specific adeno-associated virus recombinant expression vector.

[0006] This invention targets Ces1d as a regulatory marker and adeno-associated virus (AVV9) as a vector to precisely target cardiomyocytes. By increasing the expression level of Ces1d in cardiomyocytes, it significantly promotes fatty acid oxidation in cardiomyocytes, upregulates the expression of fatty acid oxidation-related genes such as Cpt1a, Cpt1b, Cpt2, Acadm, Acadvl, and Acox1, thereby optimizing the utilization of myocardial energy metabolism substrates. Simultaneously, it improves stress-induced mitochondrial dysfunction and impaired mitochondrial formation, enhances the expression of PGC1-α and oxidative respiratory chain-related genes Cycs, Cox4, Atp5b, Atp5c, and Tfam, promotes mitochondrial biosynthesis and functional recovery, improves ATP synthesis capacity, and thus improves the energy supply status of cardiomyocytes, alleviates pathological myocardial hypertrophy caused by stress, and delays its progression to heart failure. Therefore, the regulatory strategy targeting Ces1d provides a novel gene therapy approach for the prevention and treatment of myocardial hypertrophy and related cardiovascular diseases, with promising application prospects and significant clinical potential value. Attached Figure Description

[0007] Figure 1 The diagram shows the potential role of Ces1d in the development and progression of myocardial hypertrophy. A represents the number of differentially expressed genes in the myocardium of hypertrophic mice; B shows the enrichment results of fatty acid metabolism-related pathways; C is a heatmap of fatty acid metabolism-related genes; D shows myocardial CES1 expression detected by GEO data: in the GSE160997 database, n=5 normal myocardial patients and n=18 hypertrophic patients; in the GSE130036 database, n=9 normal myocardial patients and n=28 hypertrophic patients; in the GSE68316 database, n=5 normal myocardial patients and n=7 hypertrophic patients; E shows IHC detection of Ces1d protein expression in the myocardium of hypertrophic patients and mice (scale bars=20μm, n=4-5); F shows Western blot detection of Ces1d protein expression levels in cardiomyocytes (n=5-6); G shows RT-qPCR detection of Ces1d mRNA expression in the heart of mice after TAC surgery (n=6-7); H shows RT-qPCR detection of Ang II-induced Ces1d mRNA expression in the heart of mice (n=7). Figure 2 Figure 1 shows the results of myocardial hypertrophy induced by myocardial-specific overexpression of Ces1d in an in vivo model, which alleviates pressure-induced hypertrophy. A represents the HW / BW and HW / TL ratios in mice after modeling (n=5-6); B represents changes in cardiac function detected by echocardiography (n=5-6); C represents HE staining results of myocardium in mice (scale bars=50μm, n=4); D represents RT-qPCR detection of Ces1d mRNA expression in mouse myocardium (n=5-6); E represents RT-qPCR detection of markers of myocardial hypertrophy in mice (n=5-6). Figure 3 Figure 1 shows the results of specific knockdown of Ces1d in an in vitro model exacerbating AngII-induced cardiomyocyte hypertrophy. A shows the Ces1d mRNA level in primary cardiomyocytes detected by RT-qPCR (n=5); B shows the Ces1d protein expression level in cardiomyocytes detected by Western blot (n=3); C shows the Ces1d mRNA expression level in cardiomyocytes detected by RT-qPCR (n=3); D shows the cell surface area detected by CoraLite594-phalloidin staining (scale bars=20μm, n=6); E shows the mRNA level of cardiomyocyte hypertrophy markers detected by RT-qPCR (n=3). Figure 4 Figure 1 shows the results of Ces1d reducing stress-induced myocardial hypertrophy by promoting fatty acid oxidation in cardiomyocytes; where A represents the expression of key enzymes in fatty acid metabolism in mouse myocardial tissue detected by RT-qPCR, n=4~6; and B represents the expression of genes related to mitochondrial synthesis and oxidative respiratory chain in mouse myocardial tissue detected by RT-qPCR, n=4~6. Detailed Implementation

[0008] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0009] Example 1: Study on myocardial hypertrophy RNA-seq analysis was performed on mouse heart tissue 4 weeks after sham surgery and aortic arch coarctation (TAC). The results showed that 339 genes were upregulated and 262 genes were downregulated during myocardial hypertrophy. Figure 1 A). Myocardial hypertrophy is accompanied by a decrease in fatty acid oxidation, which may be related to the downregulation of transcription of certain key lipid metabolism genes. Therefore, GSEA enrichment analysis was performed on 262 downregulated genes, revealing that the changes in fatty acid oxidation-related genes were the most significant among lipid metabolism-related genes. Figure 1 B). To further clarify which key lipid metabolism genes were downregulated and caused changes in fatty acid oxidation, lipid metabolism-related genes were sorted according to their shift change (B). Figure 1(C) The expression of Aldh1l2, Pla2g5, and carboxylesterase 1 d (Ces1d) genes was significantly reduced. Aldh1l2 is a mitochondrial 10-formyltetrahydrofolate dehydrogenase that catalyzes the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 in a NADP+-dependent manner, but does not directly regulate fatty acid oxidation. Pla2g5's main function is to hydrolyze low-density lipoprotein lecithin, releasing unsaturated fatty acids. Since low-density lipoprotein is in the circulating blood, Pla2g5 cannot hydrolyze low-density lipoprotein within cardiomyocytes. Therefore, Aldh1l2 and Pla2g5 are not the initiating molecules for regulating fatty acid oxidation in cardiomyocytes. Ces1d can hydrolyze neutral lipids such as monoglycerides and triglycerides to generate free fatty acids. Its downregulation may reduce the content of free fatty acids in cardiomyocytes, thereby indirectly inhibiting fatty acid oxidation. The above results indicate that Ces1d may be an upstream molecule in the downregulation of fatty acid oxidation in cardiomyocytes.

[0010] Further analysis in the GEO database of patients with myocardial hypertrophy (GSE160997, GSE130036, GSE68316) revealed that CES1 (the human homolog of Ces1d) was significantly lower in hypertrophic myocardium compared to the control group. Figure 1 (D) This further suggests that a decrease in Ces1d plays a central role in the development and progression of myocardial hypertrophy, and may be an initiating factor rather than a difference due to species differences. Therefore, Ces1d was chosen for further research.

[0011] To further verify the changes in Ces1d expression during myocardial hypertrophy, IHC was used to detect changes in CES1 (Ces1d) protein levels in the heart tissues of patients and mice with myocardial hypertrophy. Compared with the control group, the levels of CES1 (Ces1d) protein in hypertrophic myocardium were significantly decreased. Figure 1 E). Subsequently, a pressure-load-induced myocardial hypertrophy mouse model was established, and in vivo validation revealed that the expression of Ces1d protein and mRNA in the heart of hypertrophic mice was significantly downregulated (E). Figure 1 (F, G, H), further suggesting that Ces1d may play an important role in the occurrence and development of myocardial hypertrophy.

[0012] Example 2: Exploring the function of Ces1d in myocardial hypertrophy To further explore the function of Ces1d in myocardial hypertrophy, Ces1d was overexpressed in cardiomyocytes of hypertrophic mice via tail vein injection of adeno-associated virus 9 (AVV9). The average expression level of Ces1d mRNA in the myocardium of overexpressing mice was approximately 12-fold higher than that in the control group. Figure 2D), indicating successful AVV9-Ces1d overexpression. Results showed that the heart weight to body weight ratio (HW / BW) and heart weight to tibia length ratio (HW / TL) were increased in mice with myocardial hypertrophy. Figure 2 A), left ventricular short axis shortening (FS) and left ventricular ejection fraction (EF) were significantly lower than those in the control group. Figure 2 B). Meanwhile, HE staining results of the myocardium in both groups of mice showed that the myocardial cells in the control group were uniformly stained and similar in size. In contrast, the myocardial cells in the AngII sustained-release pump-induced model mice were significantly enlarged. Figure 2 C), the expression of myocardial hypertrophy-related genes β-MHC and BNP was also significantly increased. Figure 2 E). Overexpression of Ces1d significantly reduced HW / BW and HW / TL in pressure-load-induced myocardial hypertrophy mice, and significantly improved their cardiac function and cardiomyocyte hypertrophy. Figure 2 (A, B, C, E). These results confirm that overexpression of Ces1d alleviates AngII-induced myocardial hypertrophy, and targeting Ces1d may provide a new strategy for the treatment of myocardial hypertrophy.

[0013] Next, an in vitro model was constructed using AngII intervention in primary cardiomyocytes, and it was found that Ces1d expression was also significantly reduced in hypertrophic cardiomyocytes. Figure 3 A). Subsequently, a Ces1d knockdown adenovirus (Ad-Ces1d-sh) was constructed, and the Ces1d knockdown efficiency of cardiomyocytes after adenovirus transfection was examined. It was found that Ad-Ces1d-sh1 had the highest knockdown efficiency. Figure 3 (B, C), therefore Ad-Ces1d-sh1 was used for subsequent research. The results showed that knockdown of Ces1d further aggravated AngII-induced cardiomyocyte hypertrophy (B, C). Figure 3 D), upregulates cardiac fat marker mRNA levels ( Figure 3 E). These results further confirm the protective effect of Ces1d against stress-induced myocardial hypertrophy.

[0014] To investigate the mechanism by which Ces1d alleviates stress-induced myocardial hypertrophy, a preliminary mechanistic exploration was conducted. Previous studies have suggested that Ces1d is related to lipid metabolism, implying that Ces1d may alleviate stress-induced myocardial hypertrophy by promoting fatty acid oxidation in cardiomyocytes. Therefore, after successfully modeling myocardial hypertrophy in mice, genes related to lipid metabolism in mouse myocardium were examined. It was found that the expression of key fatty acid transport enzymes Cpt2, Cpt1a and its muscle-specific subtype Cpt1b, fatty acid oxidation-related enzymes Acadm and Acadvl, and the key peroxisome β-oxidation enzyme Acox1 were significantly reduced in hypertrophic myocardium. Overexpression of Ces1d promoted the expression of Cpt1a, Cpt1b, Cpt2, Acadm, Acadvl, and Acox1. Figure 4 A), indicating that Ces1d promotes fatty acid oxidation in cardiomyocytes. We also detected mitochondrial generation-related genes PGC1-α and oxidative respiratory chain-related genes Cycs, Cox4, Atp5b, Atp5c, and Tfam (A). Figure 4 (B) It was found that the expression of genes related to mitochondrial generation and function was significantly reduced. Overexpression of Ces1d significantly alleviated the impaired mitochondrial function and generation caused by stress. These results suggest that Ces1d may alleviate stress-induced myocardial hypertrophy by promoting fatty acid oxidation in cardiomyocytes and improving mitochondrial function and generation, thereby increasing ATP synthesis capacity.

[0015] In conclusion, targeting Ces1d may be a novel approach to treating myocardial hypertrophy, providing new insights for research on the prevention and treatment of myocardial hypertrophy and even heart failure.

Claims

1. Application of the Ces1d gene as a potential therapeutic target for myocardial hypertrophy and heart failure.

2. The application as described in claim 1, characterized in that, The gene described herein has the amino acid sequence SEQ ID NO:

1.

3. The application as described in claim 1, characterized in that, The gene in question has the nucleotide sequence SEQ ID NO:

2.

4. A product for the prevention or treatment of myocardial hypertrophy and heart failure, characterized in that, The product contains a reagent that can increase the expression level of the Ces1d gene.

5. The article of claim 4, characterized in that, The product is an expression vector capable of recombinantly expressing the Ces1d gene with the amino acid sequence SEQ ID NO:

1.

6. The article of claim 5, characterized in that, The expression vector is a cardiomyocyte-specific adeno-associated virus expression vector.

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