Construction method and application of ejection fraction retention type heart failure animal model

Abstract

According to the invention, atrial fibrillation and hypertension are combined, and a novel HFpEF double-strike animal model is developed. Specifically, the animal model disclosed by the invention can stably develop to a mild structure and functional disease stage within three weeks of double strike, reaches an end stage within two subsequent weeks and gradually dies, and the animal model is highly overlapped with the pathophysiological phenotype of the existing human HFpEF. The animal model stably and rapidly develops in a staged manner, is easy to implement, and provides a valuable tool for molecular mechanism research and drug development of disease occurrence and development.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and experimental animal model design, specifically to an animal model of heart failure with preserved ejection fraction that combines the phenotypes of atrial fibrillation and hypertension. Background Technology

[0002] Heart failure (HF) is a complex clinical syndrome caused by impaired cardiac contraction or blood filling. Based on left ventricular systolic function, HF can be classified as reduced ejection fraction (HFrEF), mildly reduced ejection fraction (HFmrEF), or preserved ejection fraction (HFpEF). HFpEF accounts for 50% of all HF cases, and its prevalence increases with population aging. More than 4% of people aged 65 or older are affected by HFpEF, with a five-year survival rate of approximately 50%, similar to HFrEF. Compared to HFrEF, HFpEF has a more complex etiology, and its symptoms typically include left ventricular hypertrophy, increased left ventricular stiffness, and fluid retention. HFpEF involves multiple risk factors and multi-organ complications, and the specific molecular pathogenic mechanisms are not fully understood. Currently known drugs for treating HFrEF have not shown significant therapeutic effects on the mortality rate of HFpEF.

[0003] Because clinical tissue samples from patients with HFpEF are extremely limited, mechanistic studies primarily rely on animal models. Currently, pressure overload induced by aortic systolic artery closure (TAC) is the most widely used method for simulating ventricular hypertrophy and remodeling. However, TAC models inevitably develop into HFrEF, a phenomenon rarely observed in human patients with HFpEF. Since HFpEF is often accompanied by multiple complications such as hypertension, diabetes, obesity, coronary artery disease, chronic kidney disease, and atrial fibrillation, combining hemodynamics and metabolic stress is a commonly used modeling strategy. These include: a double-hit model of L-NAME-induced hypertension + high-fat diet (HFD); a double-hit model of mild TAC + HFD; a triple-hit SAUNA model of saline drinking water + unilateral nephrectomy + aldosterone; and a triple-hit model of HFD + deoxycorticosterone + aging (13 months). These methods provide valuable molecular insights into various pathological changes under different conditions, but these modeling strategies are usually time-consuming (4-13 months after the start of combination therapy), produce insignificant pathological phenotypes, and are generally unlikely to develop into severe late heart failure and cause death.

[0004] In summary, there is an urgent need in this field for an animal model of heart failure that preserves ejection fraction, has a short modeling time, and can produce significant pathological phenotypes. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing an animal model of heart failure with preserved ejection fraction and its application.

[0006] A first aspect of the present invention provides a method for constructing an animal model of heart failure with preserved ejection fraction, comprising the steps of:

[0007] (a) Provide an individual animal; and

[0008] (b) Inducing atrial fibrillation and hypertension in the animal individuals.

[0009] In another preferred embodiment, the animal individual is a non-human mammal, including: cattle, sheep, dogs, pigs, rabbits, non-human primates, and rodents.

[0010] In another preferred embodiment, the rodents include monkeys.

[0011] In another preferred embodiment, the rodents include mice and rats.

[0012] In another preferred embodiment, the non-human mammal is a mouse.

[0013] In another preferred embodiment, the method of inducing atrial fibrillation includes downregulating the expression of the Lkb1 gene in the atrium.

[0014] In another preferred embodiment, the downregulation includes gene knockout or gene knockdown.

[0015] In another preferred embodiment, the method for downregulating the expression of the Lkb1 gene is selected from the group consisting of:

[0016] (A1) Introduce deletion mutations or frameshift mutations into the Lkb1 gene;

[0017] (A2) Gene editing of the Lkb1 gene;

[0018] (A3) RNAi interference with the Lkb1 gene;

[0019] (A4) Any combination of A1 to A3 above.

[0020] In another preferred embodiment, the downregulation of Lkb1 expression is achieved by knocking out the Lkb1 gene through gene editing.

[0021] In another preferred embodiment, the downregulation of Lkb1 expression in the atrium is achieved by knocking out the Lkb1 gene using a tissue-specific gene editing system.

[0022] In another preferred embodiment, the tissue-specific gene editing system is selected from the group consisting of:

[0023] (B1) Atrial-specific promoter, recombinase, Lkb1 guide sequence expression cassette and reporter gene knockout system;

[0024] (B2) Atrial-specific promoter and ligand-inducible gene knockout system;

[0025] Or a combination thereof.

[0026] In another preferred embodiment, the Lkb1 gene is knocked out using the tissue-specific gene editing system by a method selected from the group consisting of:

[0027] (C1) Infect animal individuals carrying a reporter gene knockout system with a vector carrying an atrial-specific promoter, recombinase, and Lkb1 guide sequence expression cassette;

[0028] (C2) Injecting an inducer into animal individuals carrying an atrial-specific promoter and ligand inducible gene knockout system;

[0029] Or a combination thereof.

[0030] In another preferred embodiment, the atrial-specific promoter is Anf.

[0031] In another preferred embodiment, the recombinase comprises Cre recombinase.

[0032] In another preferred embodiment, the Lkb1 guide sequence expression cassette includes a promoter, an Lkb1 guide sequence, and a gRNA scaffold.

[0033] In another preferred embodiment, the promoter is the U6 promoter.

[0034] In another preferred embodiment, the nucleotide sequence of the Lkb1 guide sequence is shown in SEQ ID NO:1 and SEQ ID NO:2.

[0035] In another preferred embodiment, the vector comprises a viral vector.

[0036] In another preferred embodiment, the viral vector is an adeno-associated virus vector.

[0037] In another preferred embodiment, the adeno-associated virus vector is adenovirus-associated virus 9 (AAV9).

[0038] In another preferred embodiment, the reporter gene knockout system is Rosa26-LSL-Cas9.

[0039] In another preferred embodiment, the method of infecting the animal individual with the adenovirus-associated virus 9 includes subcutaneous injection.

[0040] In another preferred embodiment, the ligand-inducible gene knockout system is the CreERT2 system.

[0041] In another preferred embodiment, the inducer includes estrogen or estrogen analogues.

[0042] In another preferred embodiment, the inducer is an estrogen analogue.

[0043] In another preferred embodiment, the estrogen analogue is tamoxifen.

[0044] In another preferred embodiment, the tamoxifen is administered to the animal individual via intraperitoneal injection.

[0045] In another preferred embodiment, the method of inducing hypertension includes administering a vasopressor, formulation, or composition to the animal individual.

[0046] In another preferred embodiment, the method of inducing hypertension includes the continuous administration of a vasopressor drug, formulation, or composition to the animal individual.

[0047] In another preferred embodiment, the pressor drug, formulation, or composition includes: L-NAME, angiotensin II, and aldosterone.

[0048] In another preferred embodiment, the animal is continuously administered a vasopressor, formulation, or composition using a method selected from the group consisting of:

[0049] (D1) The animal individuals were continuously administered the vasopressor, formulation, or composition;

[0050] (D2) Continuously inject the vasopressor, formulation, or composition into the animal individual;

[0051] Or a combination thereof.

[0052] In another preferred embodiment, the manner of continuous administration includes continuous oral administration of a vasopressor, formulation, or composition.

[0053] In another preferred embodiment, the continuous injection method includes continuous administration via an implantable sustained-release pump.

[0054] In another preferred embodiment, the duration is 4 weeks.

[0055] A second aspect of the present invention provides a kit comprising:

[0056] (I) Gene-editing reagents that cause atrial fibrillation; and

[0057] (II) Drugs or drug combinations that cause hypertension.

[0058] In another preferred embodiment, the gene-editing agent causing atrial fibrillation includes a vector carrying a tissue-specific gene-editing system.

[0059] In another preferred embodiment, the tissue-specific gene editing system includes an atrial-specific promoter sequence, an Lkb1 guide sequence, and a gene editing system.

[0060] In another preferred embodiment, the gene-editing agent that induces atrial fibrillation also includes an inducing agent.

[0061] In another preferred embodiment, the nucleotide sequence of the Lkb1 guide sequence is shown in SEQ ID NO:1 and SEQ ID NO:2.

[0062] In another preferred embodiment, the gene editing system is selected from the group consisting of recombinase systems, CRISPR-Cas systems, or combinations thereof.

[0063] In another preferred embodiment, the recombinase system is selected from the group consisting of Cre-loxP system, Flp-FRT system, Dre-Rox system, or combinations thereof.

[0064] In another preferred embodiment, the recombinase system is the Cre-loxP system.

[0065] In another preferred embodiment, the CRISPR-Cas system is a CRISPR-Cas9 system.

[0066] In another preferred embodiment, the vector includes: a viral vector, a liposome vector, or an exosome vector.

[0067] In another preferred embodiment, the viral vector is selected from the group consisting of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or combinations thereof.

[0068] In another preferred embodiment, the liposome carrier is selected from the group consisting of lipid nanoparticles, lipid complexes, lipid polymeric complexes, or combinations thereof.

[0069] In another preferred embodiment, the liposome carrier surface has a targeting element.

[0070] In another preferred embodiment, the surface of the exosome carrier has a targeting element.

[0071] In another preferred embodiment, the targeting element specifically binds to atrial cardiomyocytes.

[0072] In another preferred embodiment, the drug or drug composition causing hypertension includes: L-NAME, angiotensin II, and aldosterone.

[0073] In another preferred embodiment, the drug or pharmaceutical composition causing hypertension further includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0074] In another preferred embodiment, the kit further includes a label or instruction manual indicating that the kit is intended for inducing atrial fibrillation and hypertension in animal individuals.

[0075] In another preferred embodiment, the animal is a non-human mammal.

[0076] In another preferred embodiment, the non-human mammals include: mice, rats, dogs, pigs, rabbits, and monkeys.

[0077] In another preferred embodiment, the non-human mammal is a mouse.

[0078] In another preferred embodiment, the label or instructions also indicate that the kit is used to prepare an animal model of heart failure with preserved ejection fraction.

[0079] In another preferred embodiment, the ejection fraction-preserving heart failure animal model has the following characteristics:

[0080] (Z1) Stroke volume decreases;

[0081] (Z2) Decreased end-diastolic diameter;

[0082] (Z3) The ratio of E wave to E' wave in mitral valve tissue Doppler is increased;

[0083] (Z4) Increased ventricular wall thickness;

[0084] (Z5) Increased heart weight;

[0085] (Z6) Increased cardiomyocyte volume;

[0086] (Z7) Increased area of ​​myocardial cell fibrosis;

[0087] (Z8) Increased lung weight;

[0088] (Z9) Increased exercise intolerance;

[0089] (Z10) Ejection fraction is constant;

[0090] (Z11) Heart rate is constant;

[0091] (Z12) Shorten the fraction constant; and

[0092] (Z13) Increased incidence of atrial fibrillation.

[0093] In another preferred embodiment, the “reduction in end-diastolic diameter” means that, compared with the end-diastolic diameter K0 of the control group animals, the end-diastolic diameter K1 of the ejection fraction-preserving heart failure animal model satisfies K1 / K0≤85%, preferably≤80%, more preferably≤75%, such as 75%-85%.

[0094] In another preferred embodiment, the “reduced stroke volume” means that, compared with the stroke volume L0 of the control group animals, the stroke volume L1 of the animal model of heart failure with preserved ejection fraction satisfies L1 / L0≤85%, preferably≤75%, more preferably≤65%, such as 65%-85%.

[0095] In another preferred embodiment, the "increased ratio of mitral valve tissue Doppler E wave to E' wave" means that, compared with the ratio M0 of mitral valve tissue Doppler E wave to E' wave in the control group animals, the ratio M1 of mitral valve tissue Doppler E wave to E' wave in the animal model of heart failure with preserved ejection fraction satisfies M1 / M0≥150%, preferably≥175%, more preferably≥200%, such as 150%-200%.

[0096] In another preferred embodiment, the "increased ventricular wall thickness" means that, compared with the ventricular wall thickness N0 of the control group animals, the ventricular wall thickness N1 of the ejection fraction-preserving heart failure animal model satisfies N1 / N0≥120%, preferably≥130%, more preferably≥135%, such as 120%-140%.

[0097] In another preferred embodiment, the “increased heart weight” means that, compared with the heart weight P0 of the control group animals, the heart weight P1 of the animal model of heart failure with preserved ejection fraction satisfies P1 / P0 ≥ 120%, preferably ≥ 140%, more preferably ≥ 160%, such as 120%-200%.

[0098] In another preferred embodiment, the "increased cardiomyocyte volume" means that, compared with the cardiomyocyte volume Q0 of the control group animals, the cardiomyocyte volume Q1 of the ejection fraction-preserving heart failure animal model satisfies Q1 / Q0≥200%, preferably≥250%, more preferably≥300%, such as 200%-350%.

[0099] In another preferred embodiment, the "increased area of ​​myocardial cell fibrosis" means that, compared with the area of ​​myocardial cell fibrosis R0 in the control group animals, the area of ​​myocardial cell fibrosis R1 in the animal model of heart failure with preserved ejection fraction satisfies R1 / R0≥350%, preferably≥400%, more preferably≥450%, such as 350%-500%.

[0100] In another preferred embodiment, the "increased lung weight" means that, compared with the lung weight S0 of the control group animals, the lung weight S1 of the animal model of heart failure with preserved ejection fraction satisfies S1 / S0≥120%, preferably≥140%, more preferably≥160%, such as 120%-165%.

[0101] In another preferred embodiment, "exacerbated exercise intolerance" means that, compared with the running distance T0 of the control group animals, the running distance T1 of the animal model with preserved ejection fraction heart failure satisfies T1 / T0≤50%, preferably≤45%, more preferably≤40%, such as 35%-50%.

[0102] In another preferred embodiment, "constant ejection fraction" means that, compared with the ejection fraction U0 of the control group animals, the ejection fraction U1 of the ejection fraction-preserving heart failure animal model satisfies 90% ≤ U1 / U0 ≤ 110%.

[0103] In another preferred embodiment, "constant heart rate" means that, compared with the heart rate V0 of the control group animals, the heart rate V1 of the ejection fraction-preserving heart failure animal model satisfies 90% ≤ V1 / V0 ≤ 110%.

[0104] In another preferred embodiment, "constant shortening fraction" means that the shortening fraction is the same as that of the control group animals.

[0105] In another preferred embodiment, the "increased incidence of atrial fibrillation" means that, compared with the incidence of atrial fibrillation W0 in the control group animals, the incidence of atrial fibrillation W1 in the animal model of heart failure with preserved ejection fraction satisfies W1 / W2≥700%, preferably≥800%, more preferably≥900%, such as 700%-1000%.

[0106] A third aspect of the present invention provides the use of the animal model of heart failure with preserved ejection fraction described in the first aspect of the present invention for the study of the mechanism of heart failure with preserved ejection fraction, and for the development of medical devices and drugs.

[0107] In another preferred embodiment, a multi-omics analysis method was used to investigate the molecular mechanisms of heart failure with preserved ejection fraction.

[0108] In another preferred embodiment, the multi-omics analysis includes transcriptomics analysis, proteomics analysis, and metabolomics analysis.

[0109] In another preferred embodiment, transcriptomic analysis was used to investigate the molecular mechanisms of heart failure with preserved ejection fraction.

[0110] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0111] Figure 1This study demonstrates the construction of AAV9 recombinant adeno-associated virus and the strategy for creating an atrial fibrillation model by specifically knocking down the Lkb1 gene in the atrial myocardium.

[0112] Figure 2 Mice subjected to atrial fibrillation and hypertension double-hit exhibited HFpEF characteristics.

[0113] Figure 2 a shows the experimental workflow. Rosa26-LSL-Cas9 knock-in mice were treated with AAV9-Anf-Cre-Lkb1 (gRNA) recombinant AAV9 virus and L-NAME, and analyses were performed at -2, 0, 1, 3, and 5 weeks.

[0114] Figure 2 b shows the ECG results from week 8.

[0115] Figure 2 c shows the changes in the incidence of atrial fibrillation (mice with irregular heartbeats or P wave disappearance for >10 seconds).

[0116] Figure 2 d shows the changes in atrial fibrillation burden (defined as atrial fibrillation time divided by total measurement time).

[0117] Figure 2 Image e shows a representative left ventricular echocardiogram (M map). The images are from 20 individual mice.

[0118] Figure 2 f-2n shows the results of echocardiographic assessments of each mouse at four time points. Among them, Figure 2 f represents heart rate (heart rate in minutes); Figure 2 g represents the ejection fraction (%). Figure 2 h represents the shortening fraction (%); Figure 2 i represents the end-diastolic diameter; Figure 2 j represents stroke volume; Figure 2 k is the ratio of the mitral valve E wave to the E' wave; Figure 2 l represents the thickness of the anterior wall of the left ventricle during systole; Figure 2 m is the thickness of the posterior wall of the left ventricle during systole; Figure 2 n simulates left ventricular mass.

[0119] Figure 2 o-2s is a parameter measured at the end of 5 weeks. Among them, Figure 2 o represents the area of ​​fibrosis (n = 5 for each group); Figure 2 p represents the cross-sectional area (CSA) of cardiomyocytes; n = 1000 per mouse, n = 4-5 hearts per group. Figure 2 q represents heart weight (HW) normalized to tibia length (TL; n = 12 per group); Figure 2r represents lung weight standardized to dry weight (LW / DW; control group, n=12 per group); Figure 2 s represents the exercise capacity of mice assessed by a treadmill test (n=12 per group).

[0120] Figure 2 t represents the mouse survival curve. Each group consisted of 20 mice, and the data are expressed as mean ± standard error of the mean (sem).

[0121] Figure 2 Analysis of variance (ANOVA) was used for f-2n, followed by multiple comparison tests. The p-values ​​indicating significance are shown in the figure. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0122] Figure 2 p-2t was performed using analysis of variance (ANOVA), followed by multiple comparison tests. The numbers in square brackets represent the p-values.

[0123] Figure 3 Transcriptomic analysis of mouse hearts at different stages of HFpEF is shown.

[0124] Figure 3 a shows the principal component analysis results of the three stages of HFpEF.

[0125] Figure 3 b shows the hierarchical clustering of differentially expressed genes.

[0126] Figure 3 c shows the gene set enrichment results for differentially expressed genes. Detailed Implementation

[0127] Through extensive and thorough research and experimentation, the inventors have pioneered a novel double-hit mouse model of atrial fibrillation (HFpEF) by combining atrial fibrillation and hypertension. Specifically, the mouse model exhibits progressive diastolic dysfunction, left ventricular hypertrophy, and decreased exercise capacity after 3 weeks of double-hit; congestive heart failure develops after 5 weeks, and the mice subsequently die. Known blood markers of human chronic heart failure are significantly elevated in the terminal phase of this model. The aforementioned mouse model highly overlaps with the existing pathophysiological phenotypes of human HFpEF. This mouse model develops stably and rapidly in stages and is easy to implement, providing a valuable tool for mechanistic research and drug development. Based on this, the present invention was completed.

[0128] It should be understood that the specific methods and experimental conditions of the invention described below in varying degrees of detail are intended to provide a substantive understanding of the invention. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0129] the term

[0130] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the scope of this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0131] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0132] As used in this article, the terms "atrial fibrillation," "AF," and "AF" are used interchangeably to refer to a common rapid heart rate disorder. The incidence of atrial fibrillation in patients with heart failure and post-excitement syndrome (HFpEF) is 15-41%. The presence of atrial fibrillation is associated with an increased risk of death in patients with heart failure. Therefore, atrial fibrillation may directly contribute to and exacerbate heart failure by interfering with normal hemodynamics.

[0133] As used in this article, the terms “HFpEF”, “heart failure with preserved ejection fraction”, “preservative HF”, and “preservative heart failure” can be used interchangeably, all referring to heart failure with preserved ejection fraction characterized by left ventricular hypertrophy, increased left ventricular stiffness, fluid retention, and various complications.

[0134] As used in this article, the term "terminal stage" refers to the later stage of a disease or physiological phenomenon. The terminal stage of a disease means that it can no longer be treated or alleviated with current medical technology and will lead to the death of the affected individual within a short period. In this study, mice that entered the terminal stage of heart failure exhibited symptoms such as diastolic dysfunction, ventricular wall thickness, left ventricular fibrosis, and increased heart weight, and died within 5 weeks.

[0135] As used in this article, the terms "adeno-associated virus" and "adenovirus-associated virus" are used interchangeably, referring to a class of single-stranded linear DNA viruses that have advantages such as low immunogenicity and long expression time, making them suitable for in vivo animal experiments.

[0136] As used in this article, the terms “gene knockout” and “knockout” are used interchangeably, referring to the introduction of double-strand breaks into a target gene, or the introduction of insertion or deletion mutations during non-homologous end joining repair, resulting in the loss of function of the target gene.

[0137] As used in this article, the terms “gene knockdown” and “knockdown” are used interchangeably, referring to the process of suppressing gene expression using inhibitors or repressive methods without affecting the gene sequence itself.

[0138] As used herein, the terms “control group animal,” “control group mouse,” and “control group” are used interchangeably and refer to the animal individuals or mice used in evaluating animal models of heart failure with preserved ejection fraction. The animal individuals or mice are wild-type animals or mice that have not undergone any treatment.

[0139] Gene editing system

[0140] The gene editing systems involved in this invention include recombinase systems and CRISPR-Cas systems. Recombinase systems utilize site-specific recombinases to mediate recombination between specific recognition sites, thereby achieving gene editing at specific sites. CRISPR-Cas systems, on the other hand, introduce double-strand breaks into the target gene using Cas nucleases, and then repair these breaks using homologous recombination, thus completing gene editing in the process.

[0141] In this invention, the recombinase system used is the Cre-loxP system. The Cre-loxP system is a site-specific gene editing system capable of performing deletion, insertion, translocation, and inversion at specific sites on DNA. This system can modify DNA in cells based on specific cell types or external stimuli, and is applicable to both eukaryotic and prokaryotic systems. Cre is a site-specific DNA recombinase that specifically recognizes loxP sites. Specifically, before gene editing using the Cre-loxP system, a loxP site needs to be placed on each side of the target gene fragment, located in the intron sequences flanking one or more consecutive exons. When Cre is present, it recognizes both loxP sites, ultimately causing sequence rearrangement between the two loxP sites, thereby completing gene editing.

[0142] As used herein, the term "ligand-induced gene knockout system" includes CreERT2, a gene editing system derived from the Cre-loxP system. This system consists of a Cre recombinase fused to the hormone-binding domain of the estrogen receptor (ER). Without the action of estrogen or estrogen analogs such as tamoxifen, Cre remains in the cytoplasm and cannot function; only when estrogen or estrogen analogs bind to the hormone-binding domain can Cre enter the nucleus and drive gene recombination, achieving time-specific gene knockout.

[0143] In this invention, mice carrying the Lkb1 gene containing the loxP site (i.e., Lkb1 mice) flox / flox Mice were mated with mice carrying the CreERT2 system to obtain Lkb1-aiKO mice. Injection of tamoxifen into these mice induced gene knockout of Cre.

[0144] In this invention, the CRISPR-Cas system used is the CRISPR-Cas9 system. Specifically, CRISPR / Cas9 contains a single-stranded guide RNA (gRNA or sgRNA) of the target gene and the Cas9 protein. The gRNA consists of crRNA and tracrRNA, wherein the crRNA contains a 20bp nucleotide sequence homologous to the target gene. By complementary pairing with the target gene, it guides the Cas9 protein to the PAM sequence near the target gene, cleaving the PAM sequence 3-4 bases upstream, inducing double-strand breaks in the cell, and causing loss of function of the target gene.

[0145] As used herein, the term "reporter knockout system" includes Rosa26-LSL-Cas9, which comprises a Cre recombinase-dependent Cas9 endonuclease, a 3X-FLAG epitope tag, and a CAG promoter-guided EGFP. Cas9 and EGFP expression is repressed by an upstream LSL (Lox-Stop-Lox) sequence. In the presence of Cre and gRNA, LSL repression is relieved, downstream Cas9 expression is activated, thereby editing the gene. Mice containing the Rosa26-LSL-Cas9 system are called Rosa26-LSL-Cas9 knock-in mice. In this invention, mice carrying the Rosa26-LSL-Cas9 system are infected with a virus carrying gRNA sequences of the Cre and Lkb1 genes. Cre is initiated, activating Cas9 expression in the Rosa26-LSL-Cas9 system. The Lkb1 gene gRNA sequence initiates expression, guiding Cas9 to target Lkb1 for gene knockout. Figure 1 ).

[0146] Tissue-specific gene editing system

[0147] To induce Lkb1 knockout and knockdown at a specific location in the atrium, this invention incorporates an atrial-specific promoter, such as Anf, into the gene editing system to express downstream genes or sequences at a specific location in the atrium. In this invention, the downstream genes or sequences include Cre recombinase, Lkb1 guide RNA, and CreERT2.

[0148] Lkb1 and methods for constructing existing animal models based on Lkb1

[0149] Lkb1 is a serine-threonine kinase that phosphorylates and activates AMPK and many other kinases important for cellular metabolism and physiology. Currently, an Lkb1-based mouse model is developed by using Lkb1... flox / flox The Lkb1 gene was constructed by crossing mice with transgenic mice containing Cre recombinase regulated by the α-MHC promoter. This method effectively knocked out the Lkb1 gene throughout the myocardium. These whole-heart cardiomyocyte-specific Lkb1 knockout mice developed spontaneous atrial fibrillation at 4 weeks of age. However, this mouse model exhibited heart failure with ventricular systolic dysfunction and decreased ejection fraction (EF), making this method unsuitable for establishing a HFpEF model. To overcome these limitations, researchers developed a novel construction method using AAV9-Anf-Cre virus (5 × 10⁻⁶ mcg / m ... 11 One genome copy was injected into 5-day-old Lkb1 puppies. flox / flox In mice, atrial-specific Lkb1 knockout (Lkb1-aKD) was used. These mice developed spontaneous atrial fibrillation starting at 6 weeks of age, but ventricular function remained unchanged. Therefore, atrial-specific Lkb1 knockout is an ideal method for establishing an atrial fibrillation-related HFpEF model.

[0150] Lkb1 Guided Sequence Expression Box

[0151] As used herein, the terms "Lkb1 guide sequence expression cassette" and "Lkb1 gRNA expression cassette" are used interchangeably and refer to a DNA sequence capable of expressing the Lkb1 guide sequence. An expression cassette typically includes components such as a promoter, a target sequence, and a terminator. In this invention, the Lkb1 guide sequence expression cassette includes a U6 promoter, an Lkb1 gRNA sequence, and a gRNA scaffold.

[0152] When designing gRNA, restriction enzyme sites need to be added to both ends of the sequence to ligate it into the expression vector. In this invention, the gRNA sequence of Lkb1 is shown in SEQ ID NO:1 and SEQ ID NO:2, where the lowercase letters in the sequence are restriction enzyme sites, and the restriction enzyme used is BsmBI.

[0153] The pressor drugs, formulations or compositions of the present invention

[0154] As used herein, the terms "L-NAME," "angiotensin II," and "aldosterone" refer to three pressor drugs. The pressor drugs, formulations, or compositions of the present invention include, but are not limited to, L-NAME, angiotensin II, and aldosterone, as well as carriers, diluents, and excipients containing the above three pressor drugs. In one embodiment, the pressor drug, formulation, or composition is drinking water containing L-NAME.

[0155] As used in this article, the term "L-NAME" is a potent inhibitor of nonspecific endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS), and long-term use of L-NAME has been shown to induce hypertension in rodents.

[0156] As used in this article, the terms “angiotensin II” and “ANG II” are used interchangeably. ANG II can promote hypertension and atherosclerosis by activating the expression of growth-promoting and pro-inflammatory genes in vascular smooth muscle cells, leading to the contraction or narrowing of the muscular walls of arteries.

[0157] As used in this article, the term "aldosterone" is a mineralocorticoid hormone regulated by angiotensin. Aldosterone enhances the reabsorption of sodium and water, thereby increasing blood volume and raising blood pressure.

[0158] Both angiotensin II and aldosterone participate in blood pressure regulation through the renin-angiotensin-aldosterone system. This system, produced by the kidneys, is a pressor regulatory mechanism that causes vascular smooth muscle contraction and water and sodium retention, thereby producing a pressor effect. Specifically, renin secreted by the kidneys breaks down angiotensinogen into angiotensin I, which is further processed into angiotensin II. Angiotensin II causes vascular smooth muscle contraction, thus increasing blood pressure. In addition, angiotensin II also stimulates the adrenal glands to release aldosterone and the hypothalamus-pituitary gland to release vasopressin. Aldosterone and vasopressin together cause the kidneys to retain sodium, and the increase in sodium in the blood leads to water retention. Aldosterone also causes the kidneys to release potassium through urine. Ultimately, this results in an increase in blood volume and blood pressure.

[0159] Therefore, biomolecules or compounds that ultimately cause pressor effects through the renin-angiotensin-aldosterone system can be used as pressor drugs, preparations or compositions in this invention, including but not limited to analogs of angiotensin II and aldosterone, or molecules or compounds that have similar or the same functions as angiotensin II and aldosterone.

[0160] The reagent kit of the present invention and its uses

[0161] This invention provides a kit comprising a gene-editing agent for inducing atrial fibrillation and a drug or drug composition for inducing hypertension. Atrial fibrillation can be induced in an animal by using the gene-editing agent to knock out the Lkb1 gene at a specific site in the atrium, and hypertension can be induced by using the drug or drug composition for inducing hypertension, thus obtaining an HFpEF animal model.

[0162] In this invention, the gene editing reagent includes a vector and a tissue-specific gene editing system. Preferably, the vector is a viral vector. The tissue-specific gene editing system is delivered into an animal using the viral vector, thereby knocking out the Lkb1 gene at a specific site in the atrium and inducing atrial fibrillation.

[0163] The medicaments or pharmaceutical compositions of the present invention that cause hypertension include vasopressor drugs, formulations, or compositions, and pharmaceutically acceptable carriers, diluents, or excipients of said vasopressor drugs, formulations, or compositions. The carriers, diluents, or excipients facilitate the ingestion of vasopressor drugs, formulations, or compositions by individual animals, thereby inducing hypertension.

[0164] The HFpEF animal model constructed using the kit of this invention can be any non-human mammal that is easy to study and has a phenotype similar to human HFpEF disease. Preferably, the HFpEF animal model is an HFpEF mouse model.

[0165] Vectors carrying tissue-specific gene editing systems

[0166] In this invention, the vector carrying the tissue-specific gene editing system can target a specific tissue, thereby delivering the tissue-specific gene editing system into the tissue cells and exerting a targeted gene editing effect.

[0167] As used in this article, the terms “viral vector,” “liposome vector,” and “exosome vector” refer to several common genetic material or delivery systems.

[0168] As used in this article, the term "viral vector" includes three commonly used delivery vectors: lentiviral vectors, adenovirus vectors, and adeno-associated virus (AAV) vectors. Lentivirals are retroviruses that use single-stranded RNA as their genome, which is reverse transcribed into cDNA and then integrated into the host cell genome. Lentivirals contain regulatory genes and can effectively infect both dividing and quiescent cells, offering advantages such as long-term stable expression of exogenous genes and higher biosafety. Adenoviruses are double-stranded DNA viruses with a wide range of infectivity and a high capacity for loading exogenous genes. AAVs are a type of single-stranded linear DNA virus. The host can only produce infectious AAVs with the assistance of helper viruses such as adenoviruses or herpesviruses. Compared to adenoviruses, AAVs have lower immunogenicity, longer expression times, and are suitable for in vivo animal experiments.

[0169] Gene editing is performed by integrating a tissue-specific gene editing system into the genome of a viral vector, or by transfecting a plasmid vector carrying a tissue-specific gene editing system into a viral vector, and then infecting animal individuals with the viral vector carrying the tissue-specific gene editing system. In this invention, the viral vector used is AAV9.

[0170] As used herein, the term "liposome carrier" is an artificial membrane, a spherical microparticle with a lipid bilayer. Hydrophilic drugs can be encapsulated in the hydrophilic interior region of the liposome, while hydrophobic drugs can be encapsulated in the hydrocarbon chain region of the lipid bilayer. Surface-modified liposome carriers can be targeted, thereby delivering tissue-specific gene-editing systems to specific tissue cells.

[0171] As used in this article, the term "exosome carrier" refers to small, membrane-bound vesicles secreted by cells. Compared to artificial membrane carriers such as liposomes, they have a more complex lipid bilayer containing various lipids, proteins, and carbohydrates, enabling them to load multiple components. They are characterized by low immunogenicity, high physicochemical stability, and high tissue penetration, making them suitable as delivery carriers. By modifying exosomes to target specific tissue cells, they can release a tissue-specific gene-editing system to perform gene editing on individual animals.

[0172] Features of the ejection fraction-preserving heart failure animal model in this invention

[0173] The animal model of heart failure with preserved ejection fraction constructed in this invention has the following characteristics: decreased stroke volume, decreased end-diastolic diameter, increased ratio of E wave to E' wave (E / E') in mitral valve tissue Doppler, increased ventricular wall thickness, increased heart weight, increased cardiomyocyte volume, increased area of ​​cardiomyocyte fibrosis, increased lung weight, aggravated exercise intolerance, constant ejection fraction, constant heart rate, constant fractional shortening, and increased incidence of atrial fibrillation.

[0174] As used in this article, the term "stroke volume" refers to the amount of blood pumped out by one ventricle after a single heartbeat, usually referring to the left ventricle. Stroke volume is affected by factors such as preload, afterload, and myocardial contractility. Preload is the intraventricular pressure at end-diastole. Excessive preload reduces myocardial contractility, thus decreasing stroke volume. Afterload is the resistance encountered during ventricular contraction. A common afterload factor is arterial blood pressure; excessively high arterial blood pressure reduces stroke volume, leading to symptoms of heart failure. The difference between end-diastolic volume and end-systolic volume is stroke volume. The size of the end-diastolic diameter indirectly reflects the size of the end-diastolic volume; therefore, a decrease in both stroke volume and end-diastolic diameter indicates the presence of heart failure symptoms in the animal model.

[0175] As used in this article, the term "E / E'" is a clinical method for assessing cardiac systolic and diastolic function. Here, E refers to the early diastolic blood flow velocity at the mitral valve tip, and E' refers to the early diastolic myocardial motion velocity at the mitral valve annulus. A high E / E' ratio indicates the presence of heart failure symptoms.

[0176] Increased ventricular wall thickness is directly related to increased myocardial cell volume and is one of the reasons for increased heart weight. A significant cause of increased ventricular wall thickness is hypertension. The three symptoms mentioned above reflect the presence of hypertension.

[0177] As used in this article, the term "myocardial fibrosis" is a process of cardiac interstitial remodeling and a symptom of a common cardiovascular disease. It is characterized by abnormal proliferation of cardiac interstitial fibroblasts in myocardial tissue, excessive accumulation of collagen fibers, a significant increase in collagen concentration and volume fraction per unit mass of myocardium, and an imbalance and abnormal distribution of different types of collagen.

[0178] Increased lung weight reflects pulmonary congestion, which is the excessive filling of pulmonary capillaries with blood, usually caused by heart failure.

[0179] Heart failure symptoms and decreased lung function can affect an animal's exercise capacity, specifically causing exercise intolerance.

[0180] As used herein, the term "fractional shortening" is used to assess myocardial contractility by measuring changes in ventricular contractility during the cardiac cycle. The constancy of fractional shortening, ejection fraction, and heart rate indicates that the animal model of this invention is a preservative form of heart failure.

[0181] The main advantages of this invention include:

[0182] (1) This invention develops a novel HFpEF double-hit mouse model by combining atrial fibrillation and hypertension. This model can stably develop to the stage of mild structural and functional diseases in a short period of time and can cause individual death, which greatly reduces the time cost of mechanism research and drug development.

[0183] (2) This invention improves the method of constructing an atrial fibrillation model by constructing AAV9-Anf-Cre-Lkb1 virus and injecting it into more readily available Rosa26-LSL-Cas9 knock-in mice to specifically knock down atrial Lkb1, thus avoiding the presence of Lkb1. flox / flox Difficulty in strain construction and Lkb1 flox / flox The difficulty in obtaining mouse models has been reduced, thus simplifying the implementation of model construction.

[0184] (3) The mouse model of the present invention has obvious diastolic dysfunction and eventually develops into congestive heart failure, which is consistent with the typical clinical features of HFpEF.

[0185] (4) This invention is the first to perform disease characterization analysis covering all stages of disease progression in an HFpEF animal model. It found that many known human chronic heart failure blood markers, such as NPPA, NPPB, LGALS3, TIMP1 and POSTN, were significantly elevated in the terminal stage of the model, supporting the clinical relevance of the mouse model of this invention to human HFpEF.

[0186] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0187] Materials and Methods:

[0188] 1. AAV9 virus packaging:

[0189] Adenovirus-associated virus 9 (AAV9)-Anf-Cre-Lkb1 (gRNA) was packaged using a triple transfection method. In short, a mixture of AAV9 Rep / Cap packaging plasmid pAAV2 / 9 (Addgene#112865), adenovirus helper plasmid pAdDeltaF6 (Addgene#112867), and a self-constructed target plasmid Anf-Cre-Lkb1 (gRNA) in equimolar ratios was transfected into HEK293T cells via PEI. Virus was harvested 72 hours post-transfection. The final AAV9 titer was determined by qPCR. The AAV stock solution was diluted with sterile saline before injection.

[0190] 2. Animal models:

[0191] All animal experiments were approved by the Westlake University Animal Ethics Committee (Protocol #21-029-SHJ). All mice used in this study were C57BL / 6J mice. Rosa26-LSL-Cas9 knock-in mice were purchased from the Jackson Laboratory (JAX 024857). Lkb1 flox / flox Mice were purchased from GemPharmatech (stock code T007821, Nanjing, China) and crossed with Anf-CreERT2 transgenic mice (provided by Zhou Bin's laboratory) to produce induced atrial-specific Lkb1 knockout mice (Lkb1-aiKO).

[0192] A mouse model of atrial fibrillation (AF) was established using the following two methods:

[0193] (1) AAV9-Anf-Cre-Lkb1 (gRNA) adeno-associated virus diluted in sterile saline was injected subcutaneously into Rosa26-LSL-Cas9 knock-in mice on day 5 (P5) after birth. The total injection volume was 50 μL and the dose was 5 × 10⁻⁶. 11 One genome copy;

[0194] (2) 100 mg / kg tamoxifen (Sigma-Aldrich T5648) dissolved in corn oil was injected intraperitoneally into Lkb1-aiKO mice on day 14 (P14) for three consecutive days.

[0195] Both methods can specifically knock down or eliminate Lkb1 in atrial cardiomyocytes. Electrocardiogram results showed that both knockout and knockdown mice developed atrial fibrillation after 6 weeks of age.

[0196] The following two methods were used to prepare hypertension models:

[0197] (1) Add L-NAME (Beyotime ST1555) at a concentration of 0.5 g / L to drinking water within a specified time period and feed the mice with this drinking water;

[0198] (2) ANG II (RWD 2004W sustained-release pump) was infused into mice at a dose of 1 μg / kg / min for 4 weeks.

[0199] Both hypertension modeling methods were used starting at 8 weeks of age in mice.

[0200] The following three methods were used to prepare HFpEF models by combining the two phenotypes of atrial fibrillation and hypertension:

[0201] (1) Atrial fibrillation was induced in Rosa26-LSL-Cas9 knock-in mice by infecting them with AAV9-Anf-Cre-Lkb1 (gRNA) recombinant adeno-associated virus, and hypertension was induced by providing drinking water containing L-NAME.

[0202] (2) Atrial fibrillation was induced in Rosa26-LSL-Cas9 knock-in mice by infecting them with recombinant adeno-associated virus (AAV9-Anf-Cre-Lkb1(gRNA), and hypertension was induced by infusing ANG II via a slow-release pump.

[0203] (3) Tamoxifen was used to induce atrial fibrillation in Lkb1-aiKO mice, and drinking water containing L-NAME was given to induce hypertension.

[0204] Example 1: Establishing an AF model

[0205] This embodiment involves the establishment of an AF model. Due to the difficulty in obtaining Lkb1... flox / flox Mice, therefore, this invention uses AAV9-Anf-Cre-Lkb1 (gRNA) virus (dose of 5 × 10⁻⁶) 11 (One genome copy) was injected into 5-day-old Rosa26-LSL-Cas9 knock-in mice to drive atrial-specific Lkb1 knockdown using the CRISPR-Cas9 system.

[0206] like Figure 1 As shown, the Anf-Cre-Lkb1 (gRNA) transfer plasmid carries the atrial-specific promoter Anf to drive the expression of Cre recombinase in postnatal atrial cardiomyocytes. This plasmid was constructed on the backbone of plasmid AAV-U6grna1-U6grna2-cTNT-Cre (Addgene#87682), in which the cTNT promoter was replaced with the Anf promoter, and an Lkb1 gRNA sequence was inserted between the second U6 promoter and the second gRNA scaffold using the SapI enzyme.

[0207] Positive: caccgGTGATGGAGTACTGCGTATG(SEQ ID NO:1)

[0208] Reverse: aaacCATACGCAGTACTCCATCACC(SEQ ID NO:2).

[0209] Following viral infection of atrial cardiomyocytes, Cre enzyme expression begins, which further activates Cas9 expression in Rosa26-LSL-Cas9 knock-in mice. Since the Anf-Cre-Lkb1 (gRNA) recombinant adeno-associated virus vector also carries a U6 promoter-driven Lkb1 gRNA expression cassette, Cas9 can edit the Lkb1 gene under the guidance of this gRNA, resulting in the deletion of the Lkb1 allele in infected atrial cardiomyocytes.

[0210] like Figure 2 As shown in a-2d, this strategy began to induce atrial fibrillation from week 6, and its incidence and severity gradually increased over time.

[0211] Example 2: Establishment of a double-hit HFpEF mouse model

[0212] This embodiment relates to the establishment of a double-hit HFpEF mouse model. Since HFpEF patients often suffer from both hypertension and atrial fibrillation, this embodiment proposes a hypothesis that the co-occurrence of atrial fibrillation and hypertension may accelerate the development of dominant HFpEF syndrome in mice.

[0213] To verify the above hypothesis, mice were divided into four groups in this embodiment: (1) atrial fibrillation; (2) treated with L-NAME from week 8 (0.5 g / L L-NAME added to drinking water); (3) AF + L-NAME; and (4) wild-type mice without any treatment, serving as the control group. Figure 2 a).

[0214] Long-axis echocardiography revealed similar cardiac structure and function to the control group, whether using L-NAME alone or AF alone. However, the combined impact of atrial fibrillation and hypertension resulted in significant diastolic dysfunction and steadily progressed to end-stage heart failure (HF) within 5 weeks of initiating dual treatment. Figure 2 e-2n). These mice exhibited a heart failure phenotype including decreased stroke volume and end-diastolic diameter, an increased ratio of E-wave to E' wave in mitral valve tissue Doppler, indicating severe diastolic dysfunction; increased ventricular wall thickness and cardiac weight, indicating ventricular hypertrophy. Histological analysis showed increased cardiomyocyte volume and increased fibrosis. Figure 2 o, 2p). Increased lung weight indicates pulmonary congestion (o, 2p). Figure 2 r). The running distance gradually shortens, indicating increased exercise intolerance. Figure 2 Most mice died within 10 weeks of initiating the dual treatment, with a median survival of 7 weeks. Figure 2 Notably, 50% of the dying mice exhibited severe edema, consistent with congestive heart failure. No decrease in ejection fraction was observed throughout the course of the disease. Furthermore, no ventricular dilation was observed in the dead mice upon necropsy.

[0215] The above findings indicate that the development of heart failure in this model is mainly caused by diastolic dysfunction, rather than systolic dysfunction or ventricular dilation, which is consistent with the typical clinical features of HFpEF.

[0216] Example 3: Transcriptomic analysis of left ventricular myocardium at different stages of HFpEF

[0217] This embodiment involves transcriptomic analysis of left ventricular myocardium at different stages of HFpEF. To investigate the molecular mechanisms of HFpEF heart failure, this embodiment performed transcriptomic analysis on mouse left ventricular tissue.

[0218] Because this model progresses steadily and rapidly, transcriptomic data from different stages of disease progression, including the terminal, near-death stage, can be collected in a relatively short time. Three mice were collected from each stage of disease progression for analysis. Stage A represents one week after the start of dual treatment, when mice are at risk of developing heart failure. Stage B represents two weeks after dual treatment, characterized by structural and functional impairments, such as hypertrophy and elevated left ventricular filling pressure. At this stage, the mice still run a distance greater than 150 meters. Stage C represents five weeks after dual treatment, where structural and functional heart disease further develops, and the running distance decreases to less than 150 meters. At this stage, one-quarter of the mice begin to die. The control group consisted of untreated wild-type mice.

[0219] Principal component analysis showed that the four groups were completely separated. Figure 3 a). Among them, phase A and phase B samples gradually moved away from the control group, while phase C samples appeared to reverse the initial changes and further deviated from the control group in the opposite direction. Differential analysis revealed five classes of genes with different expression change patterns ( Figure 3 b). Class 1 genes are gradually downregulated during disease progression and are mainly involved in the catabolism of amino acids, organic acids, and carboxylic acids. Figure 3 c). Class 2 genes were initially downregulated and then upregulated in late stage, including genes regulating inflammation and immune responses. Class 3 genes were sharply upregulated in phase A and then downregulated. These genes included antioxidant and stress response genes. Class 4 genes were gradually upregulated before phase C and then downregulated in phase C. These genes are mainly involved in energy metabolism and mitochondrial function. Class 5 genes were overexpressed only at the end of the disease, and their main function was to promote fibrosis and inflammation. Notably, established blood biomarkers for chronic heart failure, such as NPPA and NPPB (associated with volume overload and stretching), LGALS3, TIMP1, and POSTN (associated with inflammation and fibrosis), were all located in this group, validating the clinical relevance of this HFpEF mouse model.

[0220] Example 4: Different methods of inducing atrial fibrillation and hypertension resulted in similar phenotypes of HFpEF.

[0221] This embodiment involves using different methods to induce atrial fibrillation and hypertension to rule out the specific effects of viral infection treatment and inhibitor treatment, and to observe whether an HFpEF-like phenotype is produced.

[0222] To eliminate any specific effects caused by viral infection, this embodiment employs an alternative method to knock out the Lkb1 gene, namely, Lkb1... flox / flox Mice were crossed with Anf-CreERT2 mice to produce Anf-CreERT2; Lkb1 flox / floxMice (Lkb1-aiKO). Intraperitoneal injection of tamoxifen on day 14 after birth activated Cre enzyme activity, leading to a specific deletion of the Lkb1 gene in atrial cardiomyocytes. Similar to viral infection methods, this genetically induced atrial cardiomyocyte-specific Lkb1 gene deletion induced atrial fibrillation starting at 6 weeks of age. When used in combination with L-NAME, the HFpEF phenotype appeared within 5 weeks (data not shown).

[0223] To further rule out any specific effects associated with L-NAME treatment, this example employed an alternative method of inducing hypertension via angiotensin II infusion. Mice exhibited a pronounced HFpEF phenotype within 4 weeks of combined treatment with angiotensin II infusion and viral-induced atrial fibrillation (data not shown).

[0224] The above findings indicate that, regardless of the method used to induce it, the combination of atrial fibrillation and hypertension will lead to the occurrence and development of HFpEF.

[0225] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for constructing an animal model of heart failure with preserved ejection fraction, characterized in that, Including the following steps: (a) Provide an individual animal; and (b) Inducing atrial fibrillation and hypertension in the animal individuals.

2. The method as described in claim 1, characterized in that, The animal individuals are non-human mammals, including: cattle, sheep, dogs, pigs, rabbits, non-human primates, and rodents.

3. The method as described in claim 1, characterized in that, The method for inducing atrial fibrillation includes downregulating the expression of the Lkb1 gene in the atrium.

4. The method as described in claim 3, characterized in that, The downregulation includes gene knockout or gene knockdown.

5. The method as described in claim 3, characterized in that, The method for downregulating Lkb1 gene expression is selected from the following group: (A1) Introduce deletion mutations or frameshift mutations into the Lkb1 gene; (A2) Gene editing of the Lkb1 gene; (A3) RNAi interference with the Lkb1 gene; (A4) Any combination of A1 to A3 above.

6. The method as described in claim 1, characterized in that, The method of inducing hypertension includes administering a vasopressor drug, preparation, or composition to the animal individual.

7. A reagent kit, characterized in that, The kit includes: (I) Gene-editing reagents that cause atrial fibrillation; and (II) Drugs or drug combinations that cause hypertension.

8. The kit according to claim 7, characterized in that, The kit also includes a label or instructions indicating that the kit is intended to induce atrial fibrillation and hypertension in animal individuals.

9. The reagent kit as described in claim 8, characterized in that, The label or instructions also state that the kit is used to prepare an animal model of heart failure with preserved ejection fraction.

10. The use of the animal model of heart failure with preserved ejection fraction as described in claim 1, characterized in that, Used for mechanism research, medical device and drug development in heart failure with preserved ejection fraction.

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