Use of tax1bp3 in the manufacture of a product for treating, diagnosing, screening or assessing arrhythmogenic right ventricular cardiomyopathy

Abstract

The application relates to the field of medical biotechnology, in particular to application of Tax1bp3 in preparation of products for treating, diagnosing, screening or evaluating arrhythmogenic right ventricular cardiomyopathy, wherein a construction method of a mouse model of arrhythmogenic right ventricular cardiomyopathy is disclosed: a Tax1bp3 gene knockout mouse model is constructed by using a Cre / LoxP recombination system, or a Tax1bp3 gene I33 point mutation mouse model containing a c.98T>C mutation is constructed, and the Tax1bp3 gene knockout mouse model or the Tax1bp3 gene I33 point mutation mouse model is raised to be more than eight weeks old to obtain the mouse model of arrhythmogenic right ventricular cardiomyopathy, which can accurately simulate the human genetic background and highly restore the specific pathological changes and chronic disease progression of ARVC.

Description

Technical Field

[0001] This invention relates to the field of medical biotechnology, specifically to the use of Tax1bp3 in the preparation of products for the treatment, diagnosis, screening, or evaluation of arrhythmogenic right ventricular cardiomyopathy. Background Technology

[0002] Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary cardiomyopathy characterized by progressive replacement of myocardial tissue with fibrofatty tissue. It is one of the main causes of sudden cardiac death (SCD) in adolescents and athletes, accounting for 10%-20% of related sudden death cases. Its pathological manifestations include degeneration and necrosis of cardiomyocytes and subsequent repair fibrofatty infiltration, which in turn leads to ventricular structural abnormalities, thinning of the ventricular wall, and systolic dysfunction.

[0003] In the microstructure of myocardial tissue, the intercalated disc (ICD), as a specialized junctional complex between adjacent cardiomyocytes, is crucial for maintaining the mechanical coupling and electrical signal conduction of the heart. The ICD is composed of multiple junctional structures, including adhesion bands, desmosomes, and gap junctions. These structures ensure the integrity of the myocardial structure and rapid electrical conduction. Damage or remodeling of the ICD directly impairs the adhesion and electrophysiological properties between cardiomyocytes, thereby inducing severe arrhythmias and pathological cardiac remodeling. In terms of genetic mechanisms, arrhythmogenic right ventricular cardiomyopathy (ARVC) is considered a "desmosomal disease." Currently, reported ARVC pathogenic mutations are mainly concentrated in genes encoding the five core components of desmosomes, including DSP, JUP, PKP2, DSG2, and DSC2. The distribution of ARVC pathogenic mutations highlights the central role of desmosomal integrity in maintaining ICD function. These mutations weaken the mechanical connections between cardiomyocytes, making them more susceptible to damage under physiological stress and inducing inflammatory responses.

[0004] However, recent studies have shown that the genetic background of arrhythmogenic right ventricular cardiomyopathy (ARVC) is highly heterogeneous. The aforementioned classic desmosome gene variations can only explain about 50% of ARVC cases. Non-desmosome protein gene mutations also play a key role, including genes involved in calcium ion homeostasis regulation (such as RYR2), cytoskeletal protein genes (such as DES), nuclear membrane protein genes (such as LMNA), and ion channel-related genes (such as SCN5A). The role of these non-desmosome genes demonstrates that the pathogenesis of ARVC may involve multiple complex pathways such as intracellular signal transduction, metabolic homeostasis, and electrophysiological dysfunction.

[0005] Currently, the clinical treatment of ARVC focuses on symptom control and risk prevention, including limiting competitive sports, using beta-blockers and antiarrhythmic drugs, radiofrequency ablation, and implantable cardioverter-defibrillators (ICDs). However, current treatments are mostly symptomatic, lacking effective etiological treatment strategies to block or reverse the progression of myocardial fibrosis and fatty degeneration. Furthermore, the early symptoms of ARVC are often subtle and phenotypic, leading to high rates of missed and misdiagnosed cases. Many patients experience malignant arrhythmias or even sudden death at the time of initial diagnosis. Moreover, long-term use of ICDs and other devices not only carries the risk of complications but also imposes a heavy economic and psychological burden on patients. Therefore, elucidating the molecular pathogenesis of ARVC, identifying specific biomarkers for early diagnosis, and exploring novel targeted therapies to slow disease progression are of significant clinical value and scientific importance for improving patient prognosis and achieving precise prevention and control of sudden cardiac death.

[0006] Animal models are an indispensable key tool for in-depth research into the pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC) and for screening interventional drugs. By constructing animal models of arrhythmogenic right ventricular cardiomyopathy, researchers can analyze the initiation signals of myocardial remodeling at the molecular level and conduct high-throughput pharmacodynamic evaluations. Traditional animal models of arrhythmogenic right ventricular cardiomyopathy mainly rely on modifications of five desmosome genes: DSP, JUP, PKP2, DSG2, and DSC2, such as gene knockout / transgenic mice of Pkp2 or Dsg2. Although these models reproduce clinical phenotypes such as fibrofatty replacement and arrhythmias to some extent, there is still a severe lack of experimental ARVC models targeting non-desmosome genes. Existing models cannot cover more than half of the non-desmosome mutation patient population, limiting in-depth research into the heterogeneous pathogenesis of arrhythmogenic right ventricular cardiomyopathy and the development of targeted therapeutic drugs. Furthermore, existing ARVC mouse models still have key shortcomings in simulating human pathological evolution: First, existing models often show diffuse whole-heart involvement, making it difficult to reproduce the typical pathological trajectory of human ARVC, which progresses from "focal right ventricular involvement" to whole-heart involvement; second, some models have problems with high phenotypic penetrance or perinatal mortality, which seriously limits the long-term dynamic observation of the chronic progression of the disease.

[0007] The reference (Robin M. Perelli, et al. TAX1BP3 Causes TRPV4-Mediated Autosomal Recessive Arrhythmogenic Cardiomyopathy[J]. Circulation Research, 2025, 136(7):667-684.) used patient-derived induced pluripotent stem cell-derived cardiomyocytes and tamoxifen-induced cardiomyocyte-specific Tax1bp3 gene knockout mice to find that Tax1bp3 gene deficiency leads to sarcoplasmic reticulum-mediated calcium ion leakage, which in turn induces arrhythmia. However, the Tax1bp3 gene knockout mice disclosed in the reference could not recreate the phenotype of arrhythmogenic right ventricular cardiomyopathy and could not simulate the human pathological evolution.

[0008] Therefore, given the aforementioned shortcomings of traditional models, developing a novel mouse model that can accurately simulate the human genetic background and highly reproduce the specific lesions and chronic disease progression of the right ventricle is of significant scientific value for overcoming the bottlenecks in basic research and clinical translation of ARVC. Summary of the Invention

[0009] The main objective of this invention is to provide the application of Tax1bp3 in the preparation of products for the treatment, diagnosis, screening or evaluation of arrhythmogenic right ventricular cardiomyopathy, in order to solve at least one of the above-mentioned technical problems.

[0010] According to a first aspect of the present invention, a method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy is provided, comprising the following steps: Using the Cre / LoxP recombination system, Tax1bp3-flox homozygous mice were crossed with myocardial-specific Cre tool mice to screen for Tax1bp3-flox heterozygous and Cre-positive mice; Among them, the myocardial-specific Cre tool mice were selected from Xmlc2-Cre tool mice or cTNT-Cre tool mice; Tax1bp3-flox heterozygous and Cre-positive mice were crossed with Tax1bp3-flox homozygous mice, and Tax1bp3-flox homozygous and Cre-positive mice were screened to obtain Tax1bp3-flox homozygous and Cre-positive mice, thus obtaining the Tax1bp3 gene knockout mouse model. Tax1bp3 gene knockout mouse models were raised to eight weeks of age or older to obtain amoxicillin-induced right ventricular cardiomyopathy mouse models.

[0011] Specifically, when the myocardial-specific Cre tool mouse is the Xmlc2-Cre tool mouse, the genotype of Tax1bp3-flox homozygous and Cre-positive mice is Xmlc2-Cre; Tax1bp3fl / fl When the myocardial-specific Cre tool mouse is a cTNT-Cre tool mouse, the genotype of Tax1bp3-flox homozygous and Cre-positive mice is cTNT-Cre; Tax1bp3 fl / fl .

[0012] The Tax1bp3 gene is located on human chromosome 17p13.2, is approximately 5.7 kb in length, contains four exons, and encodes a 124-amino acid protein widely expressed in various human tissues, including the brain, heart, kidneys, lungs, small intestine, and skeletal muscle. From a domain analysis perspective, Tax1bp3 is primarily composed of a single PDZ domain. The PDZ domain, as a key module mediating protein-protein interactions, plays a central role in biological processes such as cell signal transduction, cell adhesion, and protein transport.

[0013] In the method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy provided by this invention, the myocardial-specific Cre tool mice used in the construction of the Tax1bp3 gene knockout mouse model are selected from Xmlc2-Cre tool mice or cTNT-Cre tool mice. This allows for the activation of the promoter and the synthesis of Cre recombinase at embryonic stage ~E7.5, the earliest stage of cardiomyocyte differentiation, to complete the knockout of the Tax1bp3 gene. The Tax1bp3 gene knockout mouse model constructed by the method provided by this invention can spontaneously form the phenotype of arrhythmogenic right ventricular cardiomyopathy, accurately reproducing the phenotype of focal fibrofatty infiltration and complex electrophysiological evolution in the right ventricle in the clinical manifestations of patients with arrhythmogenic right ventricular cardiomyopathy. This provides a key in vivo research tool for the early diagnosis and targeted drug screening of arrhythmogenic right ventricular cardiomyopathy and can be applied in screening drugs for the treatment of arrhythmogenic right ventricular cardiomyopathy.

[0014] The method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy provided by this invention is simple, the modeling system is stable, and the operation is easy.

[0015] In some implementations, Tax1bp3-flox heterozygous and Cre-positive mice and Tax1bp3-flox homozygous and Cre-positive mice are obtained by PCR identification and screening. In the PCR reaction system, the Tax1bp3 primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; The Xmlc2-Cre primer pair includes an upstream primer with the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.4; The cTNT-Cre primer pair includes an upstream primer with the nucleotide sequence shown in SEQ ID NO.5 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.6.

[0016] In some implementations, loxp sequences are inserted at both ends of the segment consisting of exons 2-4 of the Tax1bp3 gene in Tax1bp3 homozygous mice. Thus, exon2, exon3, and exon4 of the Tax1bp3 gene are selected as knockout regions, and deletion of these regions leads to loss of function of the Tax1bp3 gene in mice.

[0017] According to a second aspect of the present invention, another method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy is provided, comprising the following steps: Based on the Tax1bp3 gene information, a targeting vector with the c.98T>C mutation of the Tax1bp3 gene was constructed. The targeting vector carries the c.98T>C mutation of the Tax1bp3 gene and includes, from the 5' end to the 3' end, a 3.0kb 5' homologous arm, an FRT-PGK-Neo-polyA-FRT selection marker, exon 2 containing the I33T mutation, a 3.0kb 3' homologous arm, and an MC1-TK-polyA negative selection marker. The nucleotide sequence of the targeting vector is shown in SEQ ID NO.11. After linearizing the targeting vector, it was transfected into ES cells (embryonic stem cells), positive ES cells were screened, and positive clones with correct homologous recombination were obtained by PCR identification. Positive clones with correct homologous recombination were injected into the blastocysts of C57BL / 6J mice and transplanted into pseudopregnant female mice to obtain chimeric mice. The resulting chimeric mice were mated with Flp mice, and positive F1 generation Neo-de-homozed mice were obtained through screening. The resulting positive F1 generation Neo heterozygous mice were self-crossed, and homozygous mutant I33T mice were obtained by PCR screening. m / m Mice, i.e., a mouse model of Tax1bp3 gene I33 point mutation; A mouse model of arrhythmogenic right ventricular cardiomyopathy was obtained by raising a mouse model of the Tax1bp3 gene I33 point mutation to more than eight weeks of age.

[0018] The method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy provided by this invention results in a Tax1bp3 gene I33 point mutation mouse model, namely I33T. m / mThe mouse model effectively reproduces the phenotype of arrhythmogenic right ventricular cardiomyopathy caused by cardiomyocyte-specific Tax1bp3 conditional knockout. Furthermore, this Tax1bp3 gene I33 point mutation mouse model can realistically simulate the pathological characteristics of human ARVC, providing a valuable animal tool for subsequent gene therapy research. It can be used in screening drugs for the treatment of arrhythmogenic right ventricular cardiomyopathy.

[0019] In some implementations, the linearized targeting vector is transfected into ES cells via electrotransfection.

[0020] In some implementations, G418 (Geneticin) and ganciclovir are used to screen and identify positive ES cells, FRT-PGK-Neo-polyA-FRT is a selection marker gene expression cassette to confer G418 resistance to cells, and MC1-TK-polyA is a negative selection marker gene expression cassette.

[0021] In some implementations, the targeting vector is linearized using the SacI restriction endonuclease.

[0022] In some embodiments, in the PCR reaction system for obtaining a positive clone with correct homologous recombination by PCR identification, the identification primer pair for the 5' homologous arm includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 12 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 13, and the identification primer for the 3' homologous arm includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 14 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 15.

[0023] In some implementations, the injection method is microinjection.

[0024] In some implementations, positive F1 generation Neo-de-homozed mice and homozygous mutant I33T mice are obtained by PCR screening. m / m The identification primer pair for mice includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8.

[0025] In some embodiments, the nucleotide sequence of the 5' homologous arm of the targeting vector is shown in SEQ ID NO.9, and the nucleotide sequence of the 3' homologous arm of the targeting vector is shown in SEQ ID NO.10.

[0026] According to a third aspect of the present invention, a mouse model of arrhythmogenic right ventricular cardiomyopathy is provided, which is constructed by any of the above-described construction methods.

[0027] This invention provides a mouse model of arrhythmogenic right ventricular cardiomyopathy (ARVC) with an ARVC phenotype, including right ventricular enlargement, myocardial fibrosis, fatty infiltration, right ventricular dysfunction, and ventricular arrhythmias. The mouse model of ARVC provided by this invention provides an effective experimental tool for studying the pathological mechanism of ARVC and screening drugs, and can be used in screening drugs for the treatment of arrhythmogenic right ventricular cardiomyopathy.

[0028] The myocardial tissue phenotype of the mouse model of arrhythmogenic right ventricular cardiomyopathy (ARVC) provided by this invention shows that the deletion of the Tax1bp3 gene disrupts its normal localization in the intercalated disc, thereby impairing the integrity of intercellular connections and the orderly arrangement of myofibrils, thus clarifying that Tax1bp3 is a novel intercalated disc connection gene. Based on echocardiography, electrocardiography, and pathological examination of the mouse model of arrhythmogenic right ventricular cardiomyopathy, it was determined that the deletion of the Tax1bp3 gene in cardiomyocytes induces arrhythmogenic right ventricular cardiomyopathy, and the Tax1bp3 I33T loss-of-function mutation is an important cause of ARVC. This suggests that genetic screening based on Tax1bp3 loss-of-function mutations and targeting Tax1bp3 are potential strategies for the prevention and treatment of arrhythmogenic right ventricular cardiomyopathy. Attached Figure Description

[0029] Figure 1 This is a result figure from Example 1 of the present invention, where A represents the Tax1bp3 gene knockout mouse model (Tax1bp3...). cKO A) Schematic diagram of mouse construction, B) shows the results of PCR experiment to identify mouse genotypes, and C) shows the detection of Ctrl mice and Tax1bp3. cKO Immunoblotting results of Tax1bp3 expression in mouse hearts. D is a statistical graph of Tax1bp3 expression levels in the immunoblotting of C. Data are expressed as mean ± standard deviation. p<0.01; Figure 2 This is a diagram showing the morphological analysis and cardiac section staining analysis results from Experimental Example 1 of the present invention, where A represents Tax1bp3. cKO Overall cardiac morphology of mice and CTRL mice at embryonic stages E13.5, E14.5, E15.5, E18.5 and on day 1 after birth (P1), with B representing Tax1bp3. cKO Short-axis spectral data of the heart in mice and CTRL mice at time points E13.5, E15.5, and E18.5, where C represents Tax1bp3. cKO Statistical plots of the long axis of the heart in mice and CTRL mice at time points E13.5, E15.5, and E18.5, where D represents Tax1bp3. cKOStatistical plots of axial length ratio in mice and CTRL mice at time points E13.5, E15.5, and E18.5, where E represents Tax1bp3. cKO A comparative statistical graph showing the thickness of the left ventricle (LV), interventricular septum (IVS), anterior wall of the right ventricle (Anterior RV), and inferior wall of the right ventricle (Inferior RV) on day 1 (P1) of mice and CTRL mice. F represents Tax 1bp3. cKO Heart sections from mice and CTRL mice on day 1 (P1) after H&E staining, Oil Red O staining, Masson trichrome staining, and Von Kossa histochemical staining, along with corresponding magnified images, are shown. Data are expressed as mean ± standard deviation. p<0.05; Figure 3 This is a graph showing the results of assessing cardiac function and structural changes in Experimental Example 1 of the present invention, where A represents Tax1bp3 in an 8-week-old infant. cKO Long-axis echocardiographic images of mice and CTRL mice, showing the left ventricle (LV) and right ventricle (RV). B represents Tax1bp3 in 8-week-old mice. cKO Statistical graphs of ejection fraction (EF%), fractional shortening (FS%), left ventricular end-systolic diameter (LVIDs), left ventricular end-diastolic diameter (LVIDd), right ventricular end-systolic diameter (RVIDs), right ventricular end-diastolic diameter (RVIDd), heart weight / body weight ratio (HW / BW), and heart weight / tibia length ratio (HW / TL) in mice and CTRL mice. C represents Tax1bp3 mice at 8 weeks of age. cKO Gross specimen images and H&E stained sections of the hearts of mice and CTRL mice, D represents Tax 1bp3. cKO Oil Red O staining images of heart sections from mice and CTRL mice, along with magnified views of the right ventricle (RV), interventricular septum (IVS), and left ventricle (LV) after staining. E represents Tax 1bp3. cKO Masson trichrome staining results of heart sections from mice and CTRL mice, and magnified views of the right ventricle (RV), interventricular septum (IVS), and left ventricle (LV) after staining; Figure 4 The adult Tax1bp3 in Experimental Example 1 of this invention cKO Electrocardiogram results of mice and CTRL mice, where A represents adult Tax1bp3. cKO Representative electrocardiogram (ECG) records from mice and CTRL mice. Figure B shows the quantitative analysis results of the PR interval, QRS duration, QT interval, and heart rate (HR) from ECG A; Figure C shows the analysis results of Tax1bp3. cKORepresentative electrocardiogram records of mice and CTRL mice after isoproterenol stimulation, D is a statistical graph of the number of premature ventricular contractions occurring within 5 minutes in the C graph; Figure 5 Figure A shows the results of the Tax1bp3 gene knockout mouse model in Example 2 of this invention, where A represents the Tax1bp3 gene knockout mouse model (cTNT-Cre; Tax1bp3). fl / fl Schematic diagram of mouse model, B represents Tax1bp3 fl / fl Mice and cTNT-Cre;Tax1bp3 fl / fl Overall cardiac morphology of mice on day 1 (P1), C represents adult Tax1bp3. fl / fl Mouse and adult cTNT-Cre;Tax1bp3 fl / fl Electrocardiogram of mice; Figure 6 The figures shown are the results of Experimental Example 2 of this invention. A represents the representative Western Blot bands of N-Cadherin, β-Catenin, JUP, CONNEXIN43, Tax1bp3, and the internal control GAPDH in neonatal mouse cardiomyocytes infected with Ad-LacZ (control) or Ad-Cre (knockout) adenovirus. B shows the statistical expression of N-Cadherin in neonatal mouse cardiomyocytes infected with Ad-LacZ (control) or Ad-Cre (knockout) adenovirus. C shows the statistical expression of β-Catenin in neonatal mouse cardiomyocytes infected with Ad-LacZ (control) or Ad-Cre (knockout) adenovirus. D shows the statistical expression of JUP in neonatal mouse cardiomyocytes infected with Ad-LacZ (control) or Ad-Cre (knockout) adenovirus. E shows the expression of JUP in neonatal mouse cardiomyocytes infected with Ad-LacZ (control) or Ad-Cre (knockout) adenovirus. The expression statistics of CONNEXIN43 in neonatal mouse cardiomyocytes after adenovirus infection, F is the expression statistics of Tax1bp3 in neonatal mouse cardiomyocytes after infection with Ad-LacZ (control) or Ad-Cre (knockout) adenovirus, and G is the immunofluorescence staining results of neonatal mouse cardiomyocyte tissue sections after infection with Ad-LacZ (control) or Ad-Cre (knockout) adenovirus (white arrows indicate the location of JUP at the intercalated disc, scale bar: 100μm): JUP (red), α-actinin (green) and DAPI (blue); Figure 7 This is the Tax1bp3 gene I33 point mutation mouse model (I33T) of Example 3 of the present invention. m / m Schematic diagram of mouse model construction; Figure 8 This is a result figure for Experiment Example 3 of the present invention, where A represents WT mice and I33T mice. m / mRepresentative echocardiographic images of mice: the top row shows the long axis section of the heart; the middle and bottom rows show M-mode echocardiographic images of the right ventricle (RV) and left ventricle (LV), respectively; B represents WT mice and I33T mice. m / m Quantitative statistical results of echocardiographic parameters in mice: including left ventricular ejection fraction (LVEF%), fractional shortening (LVFS%), left ventricular end-diastolic / end-systolic diameter (LVID; d / s), and right ventricular end-diastolic / end-systolic diameter (RVID; d / s). C represents WT mice and I33T mice. m / m Representative electrocardiogram (ECG) recordings from adult mice. D shows the quantitative analysis results of PR interval, QRS duration, QT interval, and heart rate (HR) in ECG C. E shows the results of isoproterenol stimulation in WT and I33T mice. m / m Representative electrocardiogram (ECG) recordings from mice. F is a statistical graph showing the number of premature ventricular contractions (PVCs) within 5 minutes compared to graph E. G represents I33T. m / m Stained sections of mouse heart (left is a panoramic view of the heart, middle is an HE staining image, and right is a Masson staining image). Detailed Implementation

[0030] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are all commercially available.

[0031] Example 1 A method for constructing a Tax1bp3 gene knockout mouse model includes the following steps: (1) Using the Cre / LoxP recombination system, Tax1bp3-flox homozygous mice, i.e. Tax1bp3 f / f F1 generation hybrid mice were obtained by crossing with Xmlc2-Cre tool mice. The F1 generation mice were weaned at 3 weeks of age, and 0.5 cm of tissue from the tail end of the mice was cut off. 180 μL of 50 mM NaOH solution was added, and the tissue was heated at 95 °C for 30 minutes to fully lyse the tissue. 10 μL of 1 M Tris-HCl (pH=8.0) was added to neutralize the reaction system, and the mixture was thoroughly mixed to extract DNA. The genotype was identified by PCR, and Tax1bp3-flox heterozygous and Xmlc2-Cre positive mice were obtained. Tax1bp3-flox mice were obtained from the Jackson Laboratory (RRID:IMSR_JAX:036219); Xmlc2-Cre utility mice were donated by Professor Ju Chen of the University of California, San Diego. (2) Tax1bp3 heterozygous and Xmlc2-Cre positive mice were crossed with Tax1bp3-flox homozygous mice. The offspring mice were screened by PCR to obtain Tax1bp3-flox homozygous and Xmlc-Cre positive mice, which were denoted as Tax1bp3. cKO Mice, with genotype Xmlc2-Cre;Tax1bp3 fl / fl Its construction diagram is as follows: Figure 1 As shown in A; Furthermore, Tax1bp3-flox homozygous and Xmlc2-Cre negative mice were obtained through screening, namely Tax1bp3 f / f As a control group (denoted as CTRL), Tax1bp3 cKO PCR identification results of mice, CTRL mice and wild-type mice are as follows: Figure 1 As shown in B, Tax1bp3 cKO The mouse lanes contain the Floxed allele and the Xmlc2 allele. cre Allele bands.

[0032] The PCR identification kit was purchased from Thermo Fisher Scientific (DreamTaq Green PCR MasterMix (2X), Thermo, K1082); the gene identification PCR reaction system (total volume 20 μL) is shown in Table 1: Table 1

[0033] The PCR identification primer sequences are shown in Table 2: Table 2

[0034] Tax1bp3 was detected by immunoblotting. cKO Expression of the Tax1bp3 gene in the hearts of mice and CTRL mice, with β-Tubulin as an internal reference protein, results are as follows: Figure 1 As shown in C, the corresponding expression level statistics are as follows: Figure 1 As shown in D, by Figure 1 It can be seen that, compared with CTRL mice, Tax1bp3 cKO Tax1bp3 expression was specifically knocked out in mouse cardiomyocytes.

[0035] Example 2 A method for constructing a Tax1bp3 gene knockout mouse model includes the following steps: (1) Using the Cre / LoxP recombination system, Tax1bp3-flox homozygous mice were crossed with cTNT-Cre tool mice to obtain F1 generation hybrid mice. The obtained F1 generation mice were weaned at 3 weeks, and 0.5 cm of tissue from the tail end of the mice was cut off. 180 μL of 50 mM NaOH solution was added, and the tissue was heated at 95℃ for 30 minutes to fully lyse the tissue. 10 μL of 1 M Tris-HCl (pH=8.0) was added to neutralize the reaction system, and the DNA was extracted after thorough mixing. The genotype was identified by PCR to obtain Tax1bp3-flox heterozygous and cTNT-Cre positive mice. Tax1bp3-flox mice were obtained from the Jackson Laboratory (RRID:IMSR_JAX:036219); cTNT-Cre tool mice were donated by Professor Ju Chen of the University of California, San Diego. (2) Tax1bp3 heterozygous and cTNT-Cre positive mice were crossed with Tax1bp3-flox homozygous mice. The offspring mice were screened by PCR to obtain Tax1bp3-flox homozygous and cTNT-Cre positive mice with the genotype cTNT-Cre. fl / fl ; denoted as cTNT-Cre; Tax1bp3 fl / fl The mouse, its construction diagram is shown below. Figure 5 As shown in A; Furthermore, Tax1bp3-flox homozygous and cTNT-Cre negative mice were selected as the control group (denoted as Tax1bp3). fl / fl Mice).

[0036] The PCR identification kit was purchased from Thermo Fisher Scientific (DreamTaq Green PCR MasterMix (2X), Thermo, K1082); the gene identification PCR reaction system (20 μL total volume) is shown in Table 3: Table 3

[0037] The PCR identification primer sequences are shown in Table 4: Table 4

[0038] Example 3 A method for constructing a mouse model with the Tax1bp3 gene I33 point mutation includes the following steps: Based on the Tax1bp3 gene information, a 3.0kb genomic fragment upstream of the mutation site was selected as the 5' homologous arm, and a 3.0kb genomic fragment downstream of the mutation site was selected as the 3' homologous arm. The FRT-PGK-Neo-polyA-FRT positive selection marker cassette and exon 2 containing the I33T mutation were introduced into the selected linearized genomic fragment containing the 5' and 3' homologous arms. A targeting vector was constructed using infusion. The targeting vector, from the 5' end to the 3' end, contained the following in sequence: a 3.0kb 5' homologous arm (nucleotide sequence as shown in SEQ ID NO. 9), the FRT-PGK-Neo-polyA-FRT positive selection marker, exon 2 containing the I33T mutation, a 3.0kb 3' homologous arm (nucleotide sequence as shown in SEQ ID NO. 10), and the MC1-TK-polyA negative selection marker. The nucleotide sequence of the targeting vector is shown in SEQ ID NO. 11. (2) Linearize the correctly sequenced target vector using SacI restriction endonuclease: 10 μg of target vector and 50 U of SacI are added to make up to 100 μL. The vector is digested at 37°C for 4 hours. The digested product is purified and dissolved in TE buffer. The concentration is adjusted to 1 μg / μL and stored at -20°C for later use.

[0039] Transfection into ES cells: JM8A3 strain mouse ES cells (C57BL / 6N background) were cultured on MEF feeder layers. ES cells in logarithmic growth phase were harvested and resuspended in PBS to a concentration of 1×10⁻⁶. 7 cells / mL. Mix 400 μL of cell suspension with 20 μg of linearized carrier and electroporate at 250 V and 500 μF.

[0040] Screening for positive ES cells: After electroporation and culturing for 48 hours, add G418 (300 μg / mL) and ganciclovir (2 μM) for positive and negative double screening. Change the screening medium every 2 days for 7-10 days. The surviving clones are candidate positive clones.

[0041] Positive clones with correct homologous recombination were obtained by PCR identification: resistant clones were selected, genomic DNA was extracted, and long-fragment PCR was used for identification. The amplified product length was approximately 4.5-5.5 kb. The primer pair for identifying the 5' homologous arm included the upstream primer with the nucleotide sequence shown in SEQ ID NO. 12 and the downstream primer with the nucleotide sequence shown in SEQ ID NO. 13. The primer pair for identifying the 3' homologous arm included the upstream primer with the nucleotide sequence shown in SEQ ID NO. 14 and the downstream primer with the nucleotide sequence shown in SEQ ID NO. 15. The PCR identification kit used was KOD-Multi&Epi- TM(TOYOBO, Code No: KME-101).

[0042] Two positive clones with correct homologous recombination were obtained through long-fragment PCR identification. The correctly identified positive clones were expanded and cultured, with some frozen and the rest used for blastocyst injection.

[0043] Positive clones were microinjected into C57BL / 6J mouse blastocysts, and chimeric mice (F0) were obtained through embryo transfer. Chimeric mice (F0) were mated with Flp mice, and positive F1 generation Neo-de-Heterozygous mice were obtained by screening. Positive F1 generation Neo heterozygous mice were self-crossed. 0.5 cm of tissue from the tail end of the mice was harvested and lysed in 180 μL of 50 mM NaOH solution. The mixture was heated at 95°C for 30 minutes to fully lyse the tissue. 10 μL of 1 M Tris-HCl (pH=8.0) was added to neutralize the reaction mixture, and the mixture was thoroughly mixed before DNA extraction. Genotyping was performed by PCR to obtain the homozygous mutant I33T. m / m Mice, the homozygous mutant I33T m / m The mouse model is a Tax1bp3 gene I33 point mutation mouse model, and its construction diagram is shown in the figure. Figure 7 As shown; Wild-type control group WT mice were also obtained through screening.

[0044] The PCR identification kit was purchased from Thermo Fisher Scientific (DreamTaq Green PCR MasterMix (2X), Thermo, K1082); the gene identification PCR reaction system (total volume 20 μL) is shown in Table 5: Table 5

[0045] Obtain homozygous mutant I33T m / m The identification primer pairs for mice are shown in Table 6: Table 6

[0046] Experimental Example 1: Heart Development 1. For Tax1bp3 in Example 1 cKO mice (1) Morphological analysis Record Tax1bp3 of Example 1 cKO The overall cardiac morphology of mice and CTRL mice at multiple time points from the embryonic stage to day 1 postnatal, specifically E13.5, E14.5, E15.5, E18.5 during the embryonic period and day 1 postnatal (P1), is shown below. The cardiac morphology at different time points is as follows: Figure 2 As shown in A, compared to CTRL, Tax1bp3 is visible. cKO The embryonic heart showed significant right ventricular dilation and severe calcifications in the ventricular wall near the outflow tract.

[0047] Analysis was conducted based on morphological changes in the heart, by Figure 2 B in Figure 2 C and Figure 2 As can be seen in D, compared with the control group, Tax1bp3 cKO In mice, the long axis length of the heart significantly increased at time points E15.5 and E18.5, while the short axis length remained unchanged. This resulted in a significant increase in the ratio of the long axis to the short axis (axial ratio) at E15.5 and E18.5, suggesting that Tax1bp3... cKO The mouse heart tends to become elongated. By day 1 after birth, the heart... Figure 2 As can be seen in E, Tax1bp3 cKO The thickness of the anterior wall of the right ventricle in mice was significantly thinned, while the thickness of the left ventricle, interventricular septum, and inferior wall of the right ventricle did not change significantly. These results indicate that knocking out the Tax1bp3 gene in embryonic cardiomyocytes using the xmlc2-Cre tool mouse can specifically affect the development of the right ventricle, leading to abnormal heart morphology and thinning of the anterior wall of the right ventricle.

[0048] (2) Staining analysis of cardiac sections To clarify Tax1bp3 cKO Microscopic pathological changes in the mouse heart, using pathological methods to analyze P1 neonatal Tax1bp3 cKO Heart sections from mice and P1 neonatal CTRL mice were pathologically sectioned and stained for analysis. Specifically, H&E (hematoxylin and eosin) staining was used to observe myocardial structure; Oil Red O staining was used to detect lipid deposition (red); Masson's trichrome staining was used to show collagen fibrosis (red); and Von Kossa (silver nitrate) staining was used to detect calcium salt deposition (brownish-black). Figure 2 As can be seen from F, compared with the control group, Tax1bp3 cKO The mouse heart exhibited multiple structural and component abnormalities: H&E staining suggested disordered myocardial tissue structure; Masson staining confirmed significant myocardial interstitial fibrosis; Oil Red O staining showed a large amount of abnormal lipid deposition in myocardial cells; Von Kossa staining further detected obvious calcification foci.

[0049] (3) Assess cardiac function and structural changes Tax1bp3 obtained in Example 1 cKO Mice and CTRL mice were fed for eight weeks and then subjected to echocardiography.

[0050] Depend on Figure 3 As can be seen in A, compared to the control group, Tax1bp3 cKO The heart chambers of the mice were significantly dilated, especially the right ventricle (RV).

[0051] Depend on Figure 3 As can be seen in B, compared to the control group, Tax1bp3 cKO Key indicators of cardiac systolic function in mice, namely ejection fraction (EF%) and fractional shortening (FS%), were significantly reduced, indicating severe impairment of systolic function. Further analysis of chamber size revealed that Tax1bp3... cKO In mice, the left ventricular end-systolic diameter (LVIDs) was significantly increased, while the end-diastolic diameter (LVIDd) remained unchanged, consistent with weakened systolic function. The right ventricular end-systolic and end-diastolic diameters (RVIDs and RVIDd) both increased sharply, indicating severe right ventricular dilatation. Furthermore, Tax1bp3... cKO The heart weight / body weight ratio (HW / BW) and heart weight / tibia length ratio (HW / TL) in mice were significantly elevated, indicating that pathological myocardial hypertrophy had occurred.

[0052] Obtain Tax1bp3 at 8 weeks of age cKO Gross heart specimens from mice and CTRL mice, and H&E-stained heart sections, were obtained by... Figure 3 As can be seen in C, Tax1bp3 cKO In mice, obvious patchy hemorrhages or ecchymoses were visible on the surface of the heart, while the control group appeared normal. H&E staining confirmed Tax1bp3. cKO The mouse heart showed significant cardiac chamber dilation and myocardial hypertrophy, consistent with ultrasound findings.

[0053] Tax1bp3 cKO Heart sections from mice and CTRL mice were stained with Oil Red O to detect lipid deposition (red); Figure 3 As can be seen in D, Tax1bp3 cKO A large number of abnormal lipid droplets (red) were found to accumulate in the right ventricular wall of the mouse heart, while no obvious deposition was observed in the interventricular septum and left ventricular region; no similar phenomenon was observed in any region of the control group.

[0054] Tax1bp3 cKO Mice and Tax1bp3 fl / fl (CTRL) Mouse heart sections were stained with Masson's trichrome to detect collagen fibrosis (blue); Figure 3 As can be seen in E, Tax1bp3 cKO Severe collagen fiber deposition (blue) was observed in the right ventricular wall of the mouse heart, while the interventricular septum and left ventricular region were relatively normal.

[0055] Tax1bp3 raised to 8 weeks of age cKO Mice and age-matched CTRL mice underwent surface electrocardiography (ECG) testing. Figure 4 As can be seen, compared with the control group (CTRL), Tax1bp3 cKO The QRS duration in mice was significantly prolonged, suggesting intraventricular conduction disorder; however, there were no significant differences in PR interval, QT interval, and heart rate (HR) between the two groups. (Tax1bp3) cKO Adult mice exhibited severe cardiac arrhythmias. Tax1bp3 mice raised to 16 weeks of age... cKO Mice and CTRL mice were subjected to isoproterenol stimulation, followed by surface electrocardiography (ECG) analysis. Figure 4 As seen in C and 4D, compared to the control group (CTRL), Tax1bp3 cKO The incidence of premature ventricular contractions was significantly increased in mice.

[0056] 2. For Tax1bp3 in Example 2 cKO mice Record Tax 1bp3 on day 1 after birth cKO Mice and Tax1bp3 fl / fl Morphological changes in the mouse heart and electrocardiographic monitoring of arrhythmias.

[0057] Depend on Figure 5 As can be seen, TNT-Cre;Tax1bp3 fl / fl One day after birth, the mice developed right ventricular dilation and severe calcifications in the ventricular wall near the outflow tract.

[0058] For adult cTNT-Cre;Tax1bp3 fl / fl Mice and Tax1bp3 fl / fl Mice underwent surface electrocardiography (ECG) testing, and the ECG results showed cTNT-Cre;Tax1bp3 fl / fl Adult mice exhibited cardiac arrhythmias.

[0059] Example 2: Tax1bp3 is a novel intercalated disk linker gene. NMCMs (primary neonatal cardiomyocytes) derived from Taxbp3-floxed mice were infected using an adenovirus-mediated Cre recombinase system (Ad-Cre) to achieve Tax1bp3 specific knockout. An adenovirus expressing LacZ (Ad-LacZ) was used as a control. Representative Western blot bands for N-Cadherin, β-Catenin, JUP, CONNEXIN43, and Tax1bp3 in neonatal mouse cardiomyocytes infected with Ad-LacZ (control) or Ad-Cre (knockout) adenovirus are shown below. Figure 6 As shown in A, GAPDH is an internal parameter, derived from... Figure 6 A and Figure 6 The F-values ​​show that Tax1bp3 protein expression was effectively knocked out in the Ad-Cre treatment group.

[0060] Depend on Figure 6 It is evident that the loss of Tax1bp3 leads to a significant downregulation of the expression of several key intercellular junction proteins, including N-Cadherin and β-Catenin, core components of adhesion junctions, CONNEXIN43, a key protein in gap junctions, and JUP (junctional globin), which is involved in both adhesion junctions and desmosomes.

[0061] Further immunofluorescence staining of myocardial tissue sections from 8-week-old mice in Example 1 revealed that, compared to CTRL mice, Tax1bp3... cKO JUP (red) expression was significantly reduced in mouse myocardium, and its localization in intercalated discs was diffuse and irregular, lacking the typical banded structure (white arrows). Sarcomere marker α-actinin (green) staining revealed Tax1bp3. cKO Disordered arrangement of myofibrils in mouse cardiomyocytes.

[0062] Therefore, myocardial-specific Tax1bp3 loss may impair the integrity of myocardial cell connections and the orderly arrangement of myofibrils by downregulating the expression of intercellular junction proteins and disrupting their normal localization in the intercalated disc.

[0063] Experimental Example 3: Evaluation of Arrhythmogenic Right Ventricular Cardiomyopathy Phenotype To investigate whether the missense mutation in the Tax1bp3 gene c.98T>C (p.Ile33Thr, I33T), associated with familial hypertrophic right ventricular cardiomyopathy, can reproduce the arrhythmogenic right ventricular cardiomyopathy phenotype caused by cardiomyocyte-specific Tax1bp3 conditional knockout, the I33T obtained in Example 3 was used... m / m Mice and WT mice were raised to 8 weeks of age under natural growth conditions. Cardiac function and structural changes in the ventricles were assessed by echocardiography. Figure 8 A and Figure 8As can be seen from B, I33T m / m Mice exhibited significant dilation of both the left ventricle (LV) and right ventricle (RV), accompanied by impaired myocardial contractility. Quantitative analysis revealed a significant increase in both the right ventricular end-diastolic diameter (RVID;d) and end-systolic diameter (RVID;s). Although the left ventricular ejection fraction (LVEF%) did not show a statistically significant difference, the overall trend suggested impaired cardiac function.

[0064] By monitoring cardiac arrhythmias using electrocardiogram (ECG), Figure 8 C and Figure 8 As can be seen in D, the electrocardiogram (ECG) record shows I33T. m / m The mice exhibited electrophysiological abnormalities such as cardiac arrhythmia. Figure 8 E and Figure 8 As can be seen from F in I33T m / m Mice were raised to 16 weeks of age and then stimulated with isoproterenol, resulting in I33T... m / m The mice exhibited significant premature ventricular contractions.

[0065] I33T was analyzed using pathological methods. m / m Mouse hearts were subjected to pathological section staining, and gross and histological morphology of the heart was analyzed. Figure 8 As can be seen in G in I33T mice, compared to WT mice, m / m The mouse heart appeared enlarged with ventricular dilation. H&E staining visually revealed the enlarged cardiac chambers, and Masson's trichrome staining confirmed significant myocardial fibrosis.

[0066] Therefore, the c.98T>C (I33T) mutation causes cardiac dilation, arrhythmia, decreased systolic function, and fibrosis, ultimately leading to a severe ARVC phenotype. These data strongly suggest that the I33T mutation... m / m The pathogenesis of c.98T>C (p.Ile33Thr,I33T) in mice was modeled, and administration of Tax1bp3 may be a potential diagnostic indicator and treatment strategy for arrhythmogenic right ventricular cardiomyopathy.

[0067] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy, characterized in that, Includes the following steps: Tax1bp3-flox homozygous mice were crossed with myocardial-specific Cre tool mice to screen for Tax1bp3-flox heterozygous and Cre-positive mice. The myocardial-specific Cre tool mouse is selected from Xmlc2-Cre tool mouse or cTNT-Cre tool mouse; The Tax1bp3-flox heterozygous and Cre-positive mice were crossed with Tax1bp3-flox homozygous mice, and Tax1bp3-flox homozygous and Cre-positive mice were screened to obtain the Tax1bp3 gene knockout mouse model. The Tax1bp3 gene knockout mouse model was raised to eight weeks of age or older to obtain a mouse model of arrhythmogenic right ventricular cardiomyopathy.

2. The method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy according to claim 1, characterized in that, Tax1bp3-flox heterozygous and Cre-positive mice and Tax1bp3-flox homozygous and Cre-positive mice were obtained by PCR identification and screening. In the PCR reaction system, the Tax1bp3 primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; The Xmlc2-Cre primer pair includes an upstream primer with the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.4; The cTNT-Cre primer pair includes an upstream primer with the nucleotide sequence shown in SEQ ID NO.5 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.

6.

3. The method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy according to claim 1 or 2, characterized in that, The Tax1bp3-flox homozygous mice have loxp sequences inserted at both ends of the segment consisting of exons 2-4 of the Tax1bp3 gene.

4. A method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy, characterized in that, Includes the following steps: A targeting vector for the Tax1bp3 gene c.98T>C mutation was constructed, wherein the targeting vector carries the Tax1bp3 gene c.98T>C mutation, and the nucleotide sequence of the targeting vector is shown in SEQ ID NO.11; The targeting vector was linearized and transfected into ES cells. Positive ES cells were screened, and positive clones with correct homologous recombination were obtained by PCR identification. Positive clones with correct homologous recombination were injected into the blastocysts of C57BL / 6J mice to obtain chimeric mice; The chimeric mice were mated with Flp mice, and positive F1 generation Neo-de-homozed mice were obtained by screening. The positive F1 generation Neo heterozygous mice were self-crossed and screened by PCR to obtain a Tax1bp3 gene I33 point mutation mouse model. The Tax1bp3 gene I33 point mutation mouse model was raised to eight weeks of age to obtain amoxicillin-induced right ventricular cardiomyopathy mouse model.

5. The method for constructing a mouse model of arrhythmogenic right ventricular cardiomyopathy according to claim 4, characterized in that, The primer pairs used to identify positive F1 generation Neo-de-Neo heterozygous mice and Tax1bp3 gene I33 point mutation mouse models obtained by PCR screening include the upstream primer with nucleotide sequence as shown in SEQ ID NO.7 and the downstream primer with nucleotide sequence as shown in SEQ ID NO.

8.

6. A mouse model of arrhythmogenic right ventricular cardiomyopathy constructed by the construction method according to any one of claims 1-5.

7. The use of the arrhythmogenic right ventricular cardiomyopathy mouse model as described in claim 6 in screening drugs for the treatment of arrhythmogenic right ventricular cardiomyopathy.

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