Construction method for animal model of heart failure with preserved ejection fraction and use thereof

Abstract

Combining atrial fibrillation and hypertension, the present application develops a novel two-hit animal model of HFpEF. Specifically, the animal model of the present application can stably progress to a stage of mild structural and functional disease within three weeks of undergoing two hits, and reach the end stage and gradually die within the subsequent two weeks. The animal model is highly similar to the existing pathophysiological phenotype of human HFpEF. The animal model of the present application progresses stably and rapidly in stages and is easy to implement. The present application provides a valuable tool for studying the molecular mechanism of disease development and progression and for drug development.

Description

Construction methods and applications of animal models of heart failure with preserved ejection fraction Technical Field

[0001] This disclosure relates to the fields of genetic engineering and experimental animal model design, and 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 disclosure is to provide a method for constructing an animal model of heart failure with preserved ejection fraction and its application.

[0006] In one aspect, this disclosure 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;

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

[0009] In some implementations, the animal individual is a non-human mammal, including mice, rats, dogs, pigs, rabbits, and monkeys.

[0010] In some implementations, the non-human mammal is a mouse.

[0011] In some implementations, the method of inducing atrial fibrillation includes downregulating the expression of the Lkb1 gene in the atrium.

[0012] In some implementations, the downregulation includes gene knockout or gene knockdown.

[0013] In some implementations, the downregulation of Lkb1 gene expression includes:

[0014] (A1) Introduce deletion mutations or frameshift mutations into the Lkb1 gene;

[0015] (A2) Gene editing of the Lkb1 gene;

[0016] (A3) RNAi interference with the Lkb1 gene;

[0017] (A4) Any combination of A1 to A3 above.

[0018] In some implementations, the downregulation of Lkb1 expression is achieved by knocking out the Lkb1 gene through gene editing.

[0019] In some implementations, the downregulation of Lkb1 expression in the atrium is achieved by knocking out the Lkb1 gene using a tissue-specific gene editing system.

[0020] In some implementations, the tissue-specific gene editing system is selected from the group consisting of:

[0021] (B1) Atrial-specific promoter, recombinase, Lkb1 guide sequence expression cassette and reporter gene knockout system;

[0022] (B2) Atrial-specific promoter and ligand-inducible gene knockout system;

[0023] Or a combination thereof.

[0024] In some implementations, the Lkb1 gene is knocked out using the tissue-specific gene editing system selected from the group consisting of:

[0025] (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;

[0026] (C2) Injecting an inducer into animal individuals carrying an atrial-specific promoter and ligand inducible gene knockout system;

[0027] Or a combination thereof.

[0028] In some implementations, the atrial-specific promoter is Anf.

[0029] In some embodiments, the recombinase includes Cre recombinase.

[0030] In some implementations, the Lkb1 guide sequence expression cassette includes a promoter, an Lkb1 guide sequence, and a gRNA scaffold.

[0031] In some implementations, the promoter is the U6 promoter.

[0032] In some embodiments, the nucleotide sequence of the Lkb1 guide sequence is shown in SEQ ID NO:1 and SEQ ID NO:2.

[0033] In some implementations, the vector includes a viral vector.

[0034] In some implementations, the viral vector is an adeno-associated virus vector.

[0035] In some implementations, the adeno-associated virus vector is adenovirus-associated virus 9 (AAV9).

[0036] In some implementations, the reporter gene knockout system is LSL-Cas9.

[0037] In some implementations, the reporter gene knockout system is Rosa26-LSL-Cas9.

[0038] In some implementations, the method of infecting the animal individual with the adenovirus-associated virus 9 includes subcutaneous injection.

[0039] In some implementations, the ligand-inducible gene knockout system is the CreERT2 system.

[0040] In some implementations, the inducer includes estrogen or estrogen analogues.

[0041] In some implementations, the inducer is an estrogen analog.

[0042] In some implementations, the estrogen analogue is tamoxifen.

[0043] In some implementations, the tamoxifen is administered to the animal individual via intraperitoneal injection.

[0044] In some embodiments, the method of causing hypertension includes administering a vasopressor, formulation, or composition to the animal individual.

[0045] In some embodiments, the method of inducing hypertension includes the continuous administration of a vasopressor, formulation, or composition to the animal individual.

[0046] In some embodiments, the pressor drug, formulation, or composition includes: L-NAME, angiotensin II, and aldosterone.

[0047] In some embodiments, the animal individual is continuously administered a vasopressor, formulation, or composition using a method selected from the group consisting of:

[0048] (D1) The animal individuals were continuously administered the vasopressor, formulation, or composition;

[0049] (D2) Continuously inject the vasopressor, formulation, or composition into the animal individual;

[0050] Or a combination thereof.

[0051] In some embodiments, the method of continuous administration includes continuous oral administration of a vasopressor, formulation, or composition.

[0052] In some implementations, the continuous injection method includes continuous delivery via an implanted sustained-release pump.

[0053] In some implementations, the duration is 4 weeks.

[0054] In another aspect, this disclosure provides a kit comprising:

[0055] (I) Gene-editing reagents that cause atrial fibrillation; and

[0056] (II) Drugs or drug combinations that cause hypertension.

[0057] In some implementations, the gene-editing agent causing atrial fibrillation includes a vector carrying a tissue-specific gene-editing system.

[0058] In some implementations, the tissue-specific gene editing system includes an atrial-specific promoter sequence, an Lkb1 guide sequence, and a gene editing system.

[0059] In some implementations, the gene-editing reagent that induces atrial fibrillation also includes an inducer.

[0060] In some embodiments, the nucleotide sequence of the Lkb1 guide sequence is shown in SEQ ID NO:1 and SEQ ID NO:2.

[0061] In some implementations, the gene editing system is selected from the group consisting of recombinase systems, CRISPR-Cas systems, or combinations thereof.

[0062] In some embodiments, the recombinase system is selected from the group consisting of Cre-loxP system, Flp-FRT system, Dre-Rox system, or combinations thereof.

[0063] In some embodiments, the recombinase system is a Cre-loxP system.

[0064] In some implementations, the CRISPR-Cas system is a CRISPR-Cas9 system.

[0065] In some implementations, the vector includes: a viral vector, a liposome vector, and an exosome vector.

[0066] In some embodiments, the viral vector is selected from the group consisting of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or combinations thereof.

[0067] In some embodiments, the liposome carrier is selected from the group consisting of lipid nanoparticles, lipid complexes, lipid polymeric complexes, or combinations thereof.

[0068] In some implementations, the liposome carrier surface has a targeting element.

[0069] In some implementations, the surface of the exosome carrier has a targeting element.

[0070] In some implementations, the targeting element specifically binds to atrial cardiomyocytes.

[0071] In some embodiments, the drug or drug composition causing hypertension includes: L-NAME, angiotensin II, and aldosterone.

[0072] In some embodiments, the drug or pharmaceutical composition causing hypertension further includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0073] In another aspect, this disclosure provides the use of the kit described in the second aspect of the invention for inducing atrial fibrillation and hypertension in an individual animal.

[0074] In some implementations, the animal individual is a non-human mammal.

[0075] In some implementations, the non-human mammals include: mice, rats, dogs, pigs, rabbits, and monkeys.

[0076] In some implementations, the non-human mammal is a mouse.

[0077] In some implementations, it is used to create animal models of heart failure with preserved ejection fraction.

[0078] In another aspect, this disclosure provides the use of the animal model of preserved ejection fraction heart failure described in the first or third aspect of the present invention for the study of the mechanism of preserved ejection fraction heart failure, and for the development of medical devices and drugs.

[0079] It should be understood that, within the scope of this disclosure, the above-described technical features 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

[0080] Figure 1 shows 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.

[0081] Figure 2 shows that mice subjected to atrial fibrillation and hypertension double-hit exhibit HFpEF characteristics.

[0082] Figure 2a 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.

[0083] Figure 2b shows the electrocardiogram results at week 8.

[0084] Figure 2c shows the changes in the incidence of atrial fibrillation (mice with irregular heartbeats or P wave disappearance for >10 seconds).

[0085] Figure 2d shows the variation in atrial fibrillation burden (defined as atrial fibrillation time divided by total measurement time).

[0086] Figure 2e shows a representative left ventricular echocardiogram (M-map). The images are from 20 individual mice.

[0087] Figures 2f-2n show the results of echocardiographic assessments of each mouse at four time points. Figure 2f shows heart rate (heart rate per minute); Figure 2g shows ejection fraction (%); Figure 2h shows fractional shortening (%); Figure 2i shows end-diastolic diameter; Figure 2j shows stroke volume; Figure 2k shows the ratio of the mitral E wave to the E' wave; Figure 2l shows the systolic anterior wall thickness of the left ventricle; Figure 2m shows the systolic posterior wall thickness of the left ventricle; and Figure 2n shows simulated left ventricular mass.

[0088] Figures 2o-2s show the parameters measured at the end of 5 weeks. Specifically, Figure 2o shows the fibrosis area (n=5 per group); Figure 2p shows the calculated cardiomyocyte CSA (cross-sectional area; n=1000 per mouse, n=4-5 hearts per group); Figure 2q shows the heart weight (HW) normalized to tibia length (TL; n=12 per group); Figure 2r shows the lung weight normalized to dry weight (LW / DW; control group, n=12 per group); and Figure 2s shows the assessment of the mice's exercise capacity using a treadmill test (n=12 per group).

[0089] Figure 2t shows the mouse survival curves. Each group consisted of 20 mice, and the data are expressed as mean ± standard error of the mean (sem).

[0090] Figures 2f-2n were analyzed using ANOVA, followed by multiple comparison tests. The p-values ​​indicating significance are shown in the figures. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0091] Figures 2p-2t were analyzed using ANOVA, followed by multiple comparison tests. The numbers in square brackets represent the p-values.

[0092] Figure 3 shows the transcriptomic analysis of mouse hearts at different stages of HFpEF.

[0093] Figure 3a shows the principal component analysis results of the three stages of HFpEF.

[0094] Figure 3b shows the hierarchical clustering of differentially expressed genes.

[0095] Figure 3c shows the gene set enrichment results for differentially expressed genes. Detailed Implementation

[0096] 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 of this disclosure 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; all known blood markers of human chronic heart failure are significantly elevated in the terminal phase of this model. The above-mentioned mouse model highly overlaps with the existing pathophysiological phenotypes of human HFpEF. This disclosed mouse model develops stably and rapidly in a phased manner and is easy to implement, providing a valuable tool for mechanism research, medical device and drug development. Based on this, the present invention was completed.

[0097] 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.

[0098] the term

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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 disclosure, the Lkb1 guide sequence expression cassette includes a U6 promoter, an Lkb1 gRNA sequence, and a gRNA scaffold.

[0105] Gene editing system

[0106] The gene editing systems disclosed herein 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. Methods and procedures for constructing desired transgenic animal individuals using recombinase systems and / or CRISPR-Cas systems are well known to those skilled in the art.

[0107] In this disclosure, 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 targeting specific cell types or using specific 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 (Flox sequence) 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 a sequence rearrangement between the two loxP sites, thereby completing gene editing. In this disclosure, the Flox sequence is knocked into exons 2-8 of the Lkb1 gene, forming Lkb1. flox / flox Mice. Those skilled in the art are familiar with using appropriate gene editing systems and methods to knock the Flox sequence into the target location. Generally, after gene editing is completed, it is necessary to verify whether the gene editing was successful. Common verification methods include sequencing, such as Sanger sequencing and next-generation sequencing. Based on the verification results, animal individuals that have successfully undergone gene editing are selected for subsequent experimental operations.

[0108] 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 to drive gene recombination, achieving time-specific gene knockout. In this disclosure, the CreERT2 open expression frame sequence is inserted after the start codon of the Anf gene to construct Anf-CreERT2 transgenic mice. Those skilled in the art are familiar with using suitable gene editing systems and methods to knock the CreERT2 open expression frame sequence into the target location, and are familiar with methods for verifying gene editing results; based on the verification results, animal individuals that have successfully undergone gene editing are selected for subsequent experimental operations.

[0109] In this disclosure, mice carrying the Lkb1 gene containing the loxP site were mated with transgenic mice carrying the CreERT2 system to obtain Lkb1-aiKO mice. Injection of tamoxifen into these mice induced gene knockout of Cre.

[0110] In this disclosure, 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 20 bp 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.

[0111] As used herein, the term "reporter gene 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 the upstream LSL (Lox-Stop-Lox) sequence. In the presence of Cre and gRNA, LSL repression is deactivated, downstream Cas9 expression is activated, and gene editing occurs.

[0112] Mice containing the Rosa26-LSL-Cas9 system are called Rosa26-LSL-Cas9 knock-in mice. In this disclosure, Rosa26-LSL-Cas9 knock-in mice are constructed by knocking in the Rosa26 region with an LSL-Cas9 expression element. "Rosa26" is a commonly used site for targeted integration of exogenous genes into mice, characterized by its widespread and stable expression. Knocking LSL-Cas9 into other suitable locations for exogenous gene expression also falls within the scope of this invention. Those skilled in the art are also familiar with using suitable gene editing systems and methods to knock in LSL-Cas9 expression elements to target locations, and are familiar with methods for verifying gene editing results; based on the verification results, animal individuals that have successfully undergone gene editing are selected for subsequent experimental operations.

[0113] In this disclosure, mice carrying the Rosa26-LSL-Cas9 system are infected with a virus carrying gRNA sequences of the Cre and Lkb1 genes. Cre is initiated and expressed, activating the expression of Cas9 in the Rosa26-LSL-Cas9 system. The gRNA sequence of the Lkb1 gene is initiated and expressed, guiding Cas9 to target Lkb1 for gene knockout.

[0114] Tissue-specific gene editing system

[0115] To induce Lkb1 knockout and knockdown at specific sites in the atrium, this disclosure incorporates an atrial-specific promoter, such as Anf, into the gene editing system to express downstream genes or sequences at specific sites in the atrium. In this disclosure, the downstream genes or sequences include Cre recombinase, Lkb1 guide RNA, and CreERT2.

[0116] Lkb1 and methods for constructing existing animal models based on Lkb1

[0117] 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 / floxIn 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.

[0118] The vasopressor drugs, formulations or compositions disclosed herein

[0119] As used herein, the terms "L-NAME," "angiotensin II," and "aldosterone" refer to three vasopressor drugs. The vasopressor drugs, formulations, or compositions disclosed herein include, but are not limited to, L-NAME, angiotensin II, and aldosterone, as well as carriers, diluents, and excipients containing the aforementioned three vasopressor drugs. In one embodiment, the vasopressor drug, formulation, or composition is drinking water containing L-NAME.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] Therefore, biomolecules or compounds that ultimately cause pressor effects through the renin-angiotensin-aldosterone system can be used as pressor drugs, formulations or compositions in this disclosure, including but not limited to analogs of angiotensin II and aldosterone, or molecules or compounds that have similar or identical functions to angiotensin II and aldosterone.

[0125] The reagent kit and its uses disclosed herein

[0126] This disclosure provides a kit comprising a gene-editing agent for inducing atrial fibrillation and a drug or drug composition for inducing hypertension. An HFpEF animal model can be obtained by inducing atrial fibrillation in an animal individual by selectively knocking out the Lkb1 gene in the atrium using the gene-editing agent and inducing hypertension using the drug or drug composition for inducing hypertension.

[0127] In this disclosure, 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 a viral vector, thereby knocking out the Lkb1 gene at a specific site in the atrium and inducing atrial fibrillation.

[0128] The drugs or pharmaceutical compositions disclosed herein that cause hypertension include vasopressor drugs, formulations, or compositions, and pharmaceutically acceptable carriers, diluents, or excipients for 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.

[0129] The HFpEF animal model constructed using the kits disclosed herein 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.

[0130] Vectors carrying tissue-specific gene editing systems

[0131] In this disclosure, a vector carrying a tissue-specific gene editing system can target a specific tissue, thereby delivering the tissue-specific gene editing system into tissue cells and exerting a targeted gene editing effect.

[0132] As used in this article, the terms “viral vector,” “liposome vector,” and “exosome vector” refer to several common genetic material or delivery systems.

[0133] 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.

[0134] 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 an animal individual with the viral vector carrying the tissue-specific gene editing system. In this disclosure, the viral vector used is AAV9.

[0135] 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.

[0136] 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.

[0137] The main advantages of this disclosure include:

[0138] (1) This disclosure develops a novel HFpEF double-hit mouse model by combining atrial fibrillation and hypertension. This model can stably develop to a stage of mild structural and functional disease in a short period of time and can cause individual death, which greatly reduces the time cost of mechanism research and drug development.

[0139] (2) This disclosure improves the method for constructing an atrial fibrillation model by constructing an 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.

[0140] (3) The mouse model disclosed herein exhibits significant diastolic dysfunction and eventually develops into congestive heart failure, which is consistent with the typical clinical features of HFpEF.

[0141] (4) This disclosure is the first to perform disease characterization analysis covering all stages of disease progression in an animal model of HFpEF. It found that many known blood markers of human chronic heart failure, 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 disclosure to human HFpEF.

[0142] The present disclosure is 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 disclosure. 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.

[0143] Materials and Methods:

[0144] 1. AAV9 virus packaging:

[0145] Adenovirus-associated virus 9 (AAV9)-Anf-Cre-Lkb1 (gRNA) was packaged using a triple transfection method. In short, a mixture of AAV9 Rep / Cap packaged plasmid pAAV2 / 9, adenovirus helper plasmid pAdDeltaF6, and a self-constructed target plasmid Anf-Cre-Lkb1 (gRNA) was transfected into HEK293T cells using the PEI method. 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.

[0146] 2. Animal models:

[0147] 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. The LSL-Cas9 expression template was knocked into the Rosa26 region to create Rosa26-LSL-Cas9 mice. The Flox sequence was knocked into exons 2-8 of the Lkb1 gene to create Lkb1 mice. flox / flox Mice. Anf-CreERT2 transgenic mice were constructed by inserting the CreERT2 open expression cassette sequence after the start codon of the Anf gene. Lkb1 flox / flox Mice were crossed with Anf-CreERT2 mice to produce induced atrial-specific Lkb1 knockout mice (Lkb1-aiKO).

[0148] A mouse model of atrial fibrillation (AF) was established using the following two methods:

[0149] (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;

[0150] (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.

[0151] 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.

[0152] The following two methods were used to prepare hypertension models:

[0153] (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;

[0154] (2) ANG II (RWD 2004W sustained-release pump) was infused into mice at a dose of 1 μg / kg / min for 4 weeks.

[0155] Both hypertension modeling methods were used starting at 8 weeks of age in mice.

[0156] The following three methods were used to prepare HFpEF models by combining the two phenotypes of atrial fibrillation and hypertension:

[0157] (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.

[0158] (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.

[0159] (3) Tamoxifen was used to induce atrial fibrillation in Lkb1-aiKO mice, and drinking water containing L-NAME was given to induce hypertension.

[0160] Example 1: Establishing an AF model

[0161] This embodiment involves the establishment of an AF model. Due to the difficulty in obtaining Lkb1... flox / flox Mice, therefore, this disclosure discloses 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.

[0162] As shown in Figure 1, 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, 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.

[0163] Positive: caccgGTGATGGAGTACTGCGTATG(SEQ ID NO:1)

[0164] Reverse: aaacCATACGCAGTACTCCATCACC(SEQ ID NO:2).

[0165] 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.

[0166] As shown in Figures 2a-2d, this strategy began to induce atrial fibrillation from week 6, and its incidence and severity gradually increased over time.

[0167] Example 2: Establishment of a double-hit HFpEF mouse model

[0168] 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.

[0169] 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, as the control group (Figure 2a).

[0170] Long-axis echocardiography revealed that cardiac structure and function were similar to the control group, whether L-NAME or AF was used alone. However, the combined impact of atrial fibrillation and hypertension led to significant diastolic dysfunction, which steadily progressed to end-stage heart failure (HF) within 5 weeks of initiating dual treatment (Figs. 2e-2n). The HF phenotype in these mice included decreased stroke volume and end-diastolic diameter, an increased ratio of E-waves to E' waves on mitral tissue Doppler, indicating severe diastolic dysfunction, and increased ventricular wall thickness and cardiac weight, indicating ventricular hypertrophy. Histological analysis showed increased cardiomyocyte volume and exacerbated fibrosis (Figs. 2o, 2p). Increased lung weight indicated pulmonary congestion (Fig. 2r). Gradually shortened running distance reflected increased exercise intolerance (Fig. 2s). Most mice died within 10 weeks of initiating dual treatment, with a median survival of 7 weeks (Fig. 2t). 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 deceased mice during autopsy.

[0171] 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.

[0172] Example 3: Transcriptomic analysis of left ventricular myocardium at different stages of HFpEF

[0173] 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.

[0174] 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.

[0175] Principal component analysis showed complete separation of the four groups (Figure 3a). Specifically, the phase A and phase B samples gradually moved away from the control group, while the 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 patterns (Figure 3b). Class 1 genes were gradually downregulated during disease progression and were mainly involved in the catabolism of amino acids, organic acids, and carboxylic acids (Figure 3c). Class 2 genes were initially downregulated and then upregulated in later stages, 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 were mainly involved in energy metabolism and mitochondrial function. Class 5 genes were overexpressed only in the late stages 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) (18-20), were all located in this group, validating the clinical relevance of this HFpEF mouse model.

[0176] Example 4: Different methods of inducing atrial fibrillation and hypertension resulted in similar phenotypes of HFpEF.

[0177] 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.

[0178] To eliminate any specific effects caused by viral infection, this embodiment employs an alternative method to knock out the Lkb1 gene, namely, Lkb1... flox / floxMice were crossed with Anf-CreERT2 mice to produce Anf-CreERT2; Lkb1 flox / flox Mice (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).

[0179] 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).

[0180] 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.

[0181] All documents mentioned in this disclosure are incorporated herein by reference as if each document were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this disclosure, those skilled in the art can make various alterations or modifications to this disclosure, 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; (b) Inducing atrial fibrillation and hypertension in the animal individuals to obtain an animal model of heart failure with preserved ejection fraction.

2. The method as described in claim 1, characterized in that, The animal individuals are non-human mammals, including mice, rats, dogs, pigs, rabbits, and monkeys.

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 downregulation of Lkb1 gene expression includes: (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. Use of the kit according to claim 7, characterized in that, It is used to induce atrial fibrillation and high blood pressure in individual animals.

9. The use as described in claim 8, characterized in that, Used to prepare an animal model of heart failure with preserved ejection fraction.

10. The use of an animal model of heart failure with preserved ejection fraction constructed by the method of 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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