Construction method and application of spontaneous continuous ventricular tachycardia animal model

Abstract

The invention relates to a construction method and application of a spontaneous and persistent ventricular tachycardia animal model, in particular to a spontaneous and persistent ventricular tachycardia rat model based on MYL4 gene defect and myocardial infarction induction. The model is constructed by utilizing the synergistic effect of double pathological factors of MYL4 gene conserved gene defects and acquired myocardial infarction, the induction rate of the model is greater than or equal to 90%, the spontaneous duration time reaches 1-3 min, and the method is obviously superior to an existing construction method for inducing the spontaneous and continuous ventricular tachycardia model. The model can simulate the pathophysiological process of ventricular tachycardia after human MYL4 gene related cardiovascular diseases combined with myocardial infarction, provides general technical support for ventricular arrhythmia pathogenesis research, drug screening and medical instrument research and development, and has wide scientific research and clinical transformation value.

Description

Technical Field

[0001] This invention relates to the field of medical animal model technology, and in particular to a method for constructing a spontaneous sustained ventricular tachycardia animal model and its application, specifically to a method for constructing a spontaneous sustained ventricular tachycardia animal model based on MYL4 gene deficiency combined with myocardial infarction and its application. Background Technology

[0002] Ventricular tachycardia is a serious arrhythmia, especially sustained ventricular tachycardia, which can often lead to heart failure, cardiogenic shock, and even sudden death, seriously threatening human life and health. Clinically, myocardial infarction is one of the important causes of ventricular tachycardia, and genetic defects may also increase the risk of developing ventricular tachycardia. However, there are currently few studies on spontaneous sustained ventricular tachycardia induced by myocardial infarction, mainly because there is a lack of ideal animal models that can simulate this pathological process.

[0003] In the existing technology, the main methods for constructing animal models of ventricular tachycardia include drug induction, electrical stimulation, and myocardial infarction model methods. Drug-induced ventricular tachycardia (VTTA) typically uses drugs such as aconitine and ouabain. While these methods can rapidly induce VTTA, the model stability is poor and they cannot simulate the relevant pathological mechanisms. Electrical stimulation induces VTTA through external electrical stimulation of the heart, but the model duration is short, making it unsuitable for long-term research. While a simple myocardial infarction model can simulate the occurrence of arrhythmias after myocardial infarction, the induction rate is only 10% (Oknińska M, Paterek A, Bierła J, Czarnowska E, Mączewski M, Mackiewicz U. Effect of age and sex on the incidence of ventricular arrhythmia in a rat model of acute ischemia. BiomedPharmacother. 2021 Oct;142:111983.). It usually requires additional electrical stimulation to induce short runs of VT, increasing the induction rate to 50%, but the duration is still <10 s (Jiao KL, Li YG, Zhang PP, Chen RH, Yu). Y. Effects of valsartan on ventricular arrhythmia induced by programmed electrical stimulation in rats with myocardial infarction. J Cell Mol Med. 2012 Jun;16(6):1342-51.).

[0004] The MYL4 gene, located on human chromosome 17, encodes atrial myosin light chain 1, a protein crucial for cardiac development and contractile function, and is highly conserved in mammalian myocardial tissue. Recent studies have found that mutations in genes encoding myocardial structural proteins (such as MYL4) can lead to serious arrhythmias by altering cardiac electrophysiological characteristics. However, whether these mutations are related to spontaneous sustained ventricular tachycardia remains unknown, and there are currently no reports of combining MYL4 gene mutations with myocardial injury modeling to construct animal models.

[0005] Therefore, constructing a high-induction spontaneous sustained ventricular tachycardia animal model with dual pathological factors of congenital genetic defects combined with acquired myocardial infarction, and simultaneously developing a standardized and highly stable arrhythmia model construction method that can be applied across species, is of great significance for in-depth research on the pathogenesis of related arrhythmias and the development of effective therapeutic drugs and medical devices. Summary of the Invention

[0006] To overcome at least one deficiency in the existing technology, such as the inability of existing animal models of ventricular tachycardia to stably simulate continuous spontaneous ventricular tachycardia, this invention provides an animal model of spontaneous continuous ventricular tachycardia induced by MYL4 gene deficiency combined with myocardial infarction, its construction method and application, which greatly improves the stability and success rate of constructing animal models of continuous ventricular tachycardia.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention is to provide a method for constructing an animal model of cardiac arrhythmia, comprising the steps of: Step A: Select conserved arrhythmia-related genes based on the target species, and construct and select homozygous animal models with gene defects; Step B: Construct an animal model of myocardial injury using the homozygous gene defect animal model obtained in Step A; Step C: Screen animal models of cardiac arrhythmia that meet the criteria through phenotypic monitoring; The arrhythmia animal models include atrial fibrillation arrhythmia animal models, ventricular fibrillation arrhythmia animal models, spontaneous sustained ventricular tachycardia animal models, bradycardia animal models, and atrioventricular reentrant tachycardia animal models.

[0008] Furthermore, the conserved arrhythmia-related genes include SCN5A (sodium channel): long QT syndrome, Brugada syndrome, conduction block; KCNQ1, KCNH2 (potassium channels): long QT syndrome; CACNA1C (calcium channel): Timothy syndrome; GJA1 (connector protein Cx43): ischemic and heart failure arrhythmias; RYR2, CASQ2 (calcium processing protein): catecholamine-sensitive ventricular tachycardia (CPVT); MYL4 (myocardial contractility): familial hereditary atrial cardiomyopathy and atrial fibrillation arrhythmias.

[0009] Furthermore, the animal model of myocardial injury is constructed using one of the following methods: permanent coronary artery ligation model construction; drug / chemical induction model construction; electrical stimulation induction model construction; ischemia / reperfusion injury model construction; physical / mechanical injury model construction; or genetic engineering model construction.

[0010] Furthermore, the animal models of arrhythmia include: ischemia / reperfusion (I / R) induced ischemia / reperfusion arrhythmia models; permanent coronary artery ligation (MI) induced post-infarction arrhythmia models; pressure overload (such as TAC) induced hypertrophy / heart failure related arrhythmia models; and toxin-induced (such as isoproterenol) induced drug / toxicity related arrhythmia models.

[0011] The above-mentioned permanent coronary artery ligation model is constructed by surgically ligating the left anterior descending coronary artery (LAD) of a live animal (most commonly rats or mice) with sutures. For example, the heart is exposed by opening the chest, and a permanent ligation is made and knotted with sutures below the origin of the LAD (usually 1-2 mm from the root of the aorta). Then the chest is closed to simulate acute myocardial infarction without reperfusion therapy and its long-term natural outcome (heart failure, ventricular remodeling, post-infarction ventricular arrhythmia).

[0012] The construction of the aforementioned drug / chemical induction models utilizes specific chemical substances to interfere with ion channels or cause myocardial damage to induce arrhythmias. For example: Aconitine-induced model: primarily used to simulate premature ventricular contractions, ventricular tachycardia, and ventricular fibrillation. Barium chloride (BaCl2)-induced model: used to induce ventricular arrhythmias, the mechanism being the blocking of potassium channels. Calcium chloride-acetylcholine (CaCl2-Ach) mixed model: commonly used to establish atrial fibrillation (AF) or atrial flutter models. Ouabain-induced model: used to simulate ventricular arrhythmias caused by digitalis poisoning.

[0013] The above-mentioned electrical stimulation-induced models are constructed by altering cardiac electrophysiological characteristics through exogenous current. For example, the rapid atrial / ventricular pacing model induces chronic atrial fibrillation or heart failure with arrhythmias through long-term high-frequency stimulation. The programmed electrical stimulation (PES) model simulates clinical electrophysiological examinations to induce reentrant arrhythmias (such as ventricular tachycardia).

[0014] The above-mentioned ischemia / reperfusion injury models are constructed to simulate the clinicopathological states associated with coronary artery disease and myocardial infarction. For example, the coronary artery ligation model induces acute or chronic ischemic ventricular arrhythmias by ligating the coronary arteries (mostly the left anterior descending artery) and subsequent reperfusion.

[0015] The construction of the above physical / mechanical damage models includes: Atrioventricular block model: established through physical disruption (such as radiofrequency ablation, formaldehyde injection) of the His bundle or atrioventricular node. For example, aseptic pericarditis model: the pericardium is rubbed with talcum powder to study inflammation-related atrial fibrillation.

[0016] The above-mentioned genetic engineering model construction: transgenic mouse model: such as knockout or mutation of ion channel genes such as SCN5A and KCNQ1, to simulate hereditary arrhythmias such as long QT syndrome (LQTs) and Brugada syndrome.

[0017] Furthermore, the steps for constructing the spontaneous sustained ventricular tachycardia animal model include: S1. Construct animal models of MYL4 gene defect, breed and raise animals with homozygous MYL4 gene defect, and select animal models of MYL4 gene defect. S2. Using the MYL4 gene defect homozygous animal model obtained in step S1, a myocardial infarction animal model was constructed by ligation of the left anterior descending coronary artery. S3. After constructing the animal model of myocardial infarction as described in step S2, long-term electrocardiogram monitoring is performed on the animals, and the animal model of spontaneous sustained ventricular tachycardia induced by MYL4 gene deficiency combined with myocardial infarction is screened based on the monitoring results.

[0018] Further, the animals include rodents (such as mice and rats), lagomorphs (such as rabbits), canines (such as beagles), and suidae (such as miniature pigs); preferably, the animals are rodents, and more preferably, the animals are rats.

[0019] Further, the animal is a rat, specifically an SPF-grade SD strain rat; in step S1, the MYL4 gene defective animal model is a MYL4 gene defective rat model, which includes MYL4 gene knockout rats and MYL4 functional defective rats caused by gene editing.

[0020] Further, in step S1, the MYL4 gene defect homozygous rat model uses a homozygous MYL4p.E11K point mutation rat model. The construction steps are based on Chinese patent CN111621500B (application number CN202010479394.9), and the WT rats used are SPF-grade SD rats. A brief description is as follows: Glutamic acid (Glu / E), the 11th amino acid of exon 1 of the MYL4 gene in wild-type rats, is selected as the target site. An sgRNA targeting this target site and a single-stranded nucleus from rats containing the MYL4p.E11K point mutation are designed. nucleotide template (ssODN); sgRNA, ssODN and Cas9 mRNA were co-injected into one-cell stage rat zygotes via microinjection, and the injected zygotes were transplanted into pseudopregnant rats; 7 days after the birth of F0 generation rats, rat toes were harvested to extract genomes; PCR amplification primers were designed upstream and downstream of the target site so that the target sequence was included in the PCR amplification product, and identification was performed by comparison with wild-type rat genomes; MYL4p.E11K point mutant rats identified as positive were selected for breeding, and finally homozygous MYL4p.E11K point mutant rat models were obtained.

[0021] Furthermore, in step S1, the MYL4 gene expression level in the MYL4 gene-deficient homozygous rat model decreased by more than 90% compared with the wild type, meeting the conditions for inducing ventricular tachycardia after myocardial infarction.

[0022] Further, in step S2, healthy homozygous MYL4 gene-deficient rats aged 8-10 weeks and weighing 250-300g were selected and subjected to surgery after one week of acclimatization. The selection criteria for these rats were: rats at this stage have mature cardiac development, and their coronary artery diameter (approximately 0.3-0.5 mm) is suitable for 6-0 sutures; a weight of 250-300g can reduce the mortality rate during anesthesia and surgery (<5%).

[0023] Further, in step S2, the steps for constructing the rat model of myocardial infarction include: after anesthetizing the rats (by intraperitoneal injection of 6 mL / kg of 2.5% tribromoethanol), fixing them supine on the operating table, making a midline incision in the neck, separating and intubating the trachea, and connecting them to a small animal ventilator (specific parameters are a respiratory rate of 80 breaths / minute and a tidal volume of 10 mL / kg); making an incision in the 4th-5th intercostal space on the left side of the chest, separating the muscles (pectoralis major and pectoralis minor) layer by layer, opening the thoracic cavity (using a rib retractor) to expose the heart; (under a surgical microscope) locating the left anterior descending coronary artery between the left atrial appendage and the pulmonary conus, and using atraumatic cotton sutures (6-0 atraumatic cotton sutures) near the main trunk of the left coronary artery (approximately 2 km from the main trunk of the left coronary artery). The ligation was performed at a distance of mm. Successful ligation was indicated by the observation of the anterior wall of the left ventricle turning pale and the myocardial movement weakening (indicating successful establishment of the myocardial infarction model). The thoracic cavity was closed layer by layer (air was expelled from the thoracic cavity during the closure process). After the rat's spontaneous breathing was restored, the endotracheal tube was removed. Postoperative infection prevention was necessary (specifically, the rat was placed in a warm environment for recovery after surgery, and penicillin (200,000 U / kg) was administered intraperitoneally once a day for 3 consecutive days to prevent infection).

[0024] Further, in step S3, the screening criteria are as follows: 1-4 weeks post-surgery, under anesthesia (each time isoflurane-induced anesthesia), monitoring for 3 minutes, if there are ≥3 consecutive premature ventricular contractions lasting ≥30 seconds, it is judged as spontaneous sustained ventricular tachycardia.

[0025] Furthermore, the construction method also includes step S4, which involves validating and analyzing the obtained spontaneous sustained ventricular tachycardia animal model, including functional validation and / or pathological validation.

[0026] Furthermore, the functional verification includes echocardiographic detection of left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDD), and left ventricular end-systolic diameter (LVESD).

[0027] Furthermore, in the constructed rat model of spontaneous sustained ventricular tachycardia, the left ventricular ejection fraction was significantly reduced, while the left ventricular end-diastolic diameter and left ventricular end-systolic diameter were significantly increased, indicating that there was significant cardiac dysfunction.

[0028] Furthermore, the pathological verification includes HE staining to observe myocardial necrosis and Masson staining to detect fibrosis.

[0029] Furthermore, in the constructed rat model of spontaneous sustained ventricular tachycardia, a large amount of collagen fiber deposition was observed in the myocardial interstitium, indicating that there were significant myocardial damage and fibrotic changes.

[0030] Furthermore, the validation analysis in step S4 also includes gene expression detection, wherein the genes include the MYL4 gene and / or related arrhythmia regulatory genes (such as KCNQ1, SCN5A, CACNA1C) in rat myocardial tissue.

[0031] Furthermore, MYL4 gene deficiency affects the expression of ion channel-related genes in cardiomyocytes; in a rat model of spontaneous sustained ventricular tachycardia, the expression levels of Kcnd2, kcnj8, kcnd3, SCN5A, RYR2, and ATP2A2 genes in the cardiac conduction tissue were significantly decreased.

[0032] Furthermore, the success rate of constructing the spontaneous sustained ventricular tachycardia rat model is ≥80%, and the arrhythmia induction rate is ≥90%, of which ventricular tachycardia accounts for 80%.

[0033] Furthermore, the modeling time for the spontaneous sustained ventricular tachycardia rat model is 1-4 weeks. Specifically, after 1 week of modeling, some rats exhibit obvious spontaneous ventricular tachycardia; after 2 weeks, 90% of the model rats exhibit obvious spontaneous ventricular tachycardia. It is understood that, based on the high conservation of the MYL4 gene in mammalian myocardial tissue, the pathophysiological mechanism of spontaneous sustained ventricular tachycardia induced by MYL4 gene deficiency combined with myocardial infarction, as described above, is conserved across different mammals. That is, the method for constructing the spontaneous sustained ventricular tachycardia rat model described above is also applicable to other non-human mammals. Those skilled in the art can routinely adjust specific operational parameters (such as anesthetic dosage, surgical route, ligation location, and suture specifications) according to the physiological and anatomical structure of the target animal (such as body weight, heart size, and coronary artery distribution).

[0034] The second aspect of the present invention is to provide an application of the construction method as described in any of the first aspects of the present invention, or an animal model of arrhythmia constructed therefrom, selected from at least one of the following applications: the application of the animal model of arrhythmia in determining the molecular mechanism by which gene defects and myocardial injury synergistically induce arrhythmias; the application of the animal model of arrhythmia in screening antiarrhythmic drugs (evaluating the effect of drugs on the frequency, duration and cardiac function of arrhythmia attacks through phenotypic monitoring, such as a comparative study of the conversion efficacy of amiodarone vs. nifekalan for ventricular arrhythmias); and the application of the animal model of arrhythmia in the preparation of medical devices for the treatment of arrhythmias (e.g., developing a cardiac defibrillator and verifying the efficacy and safety of the device through phenotypic monitoring).

[0035] Furthermore, the arrhythmia animal model is a rat model of spontaneous sustained ventricular tachycardia induced by MYL4 gene deficiency combined with myocardial infarction. It can be used to determine the pathogenesis of ventricular tachycardia induced by MYL4 gene deficiency combined with myocardial infarction, screen drugs for treating the disease, and develop related medical devices.

[0036] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects: (1) This invention is the first to combine MYL4 gene deficiency with myocardial infarction. Compared with the simple myocardial infarction model (induction rate 20%-30%), the rat model constructed by this invention has a ventricular tachycardia induction rate ≥90%, and can spontaneously and continuously occur (1~3min) without electrical stimulation, which is more in line with the human pathological process. It can more realistically simulate the pathophysiological process of ventricular tachycardia induced by human MYL4 gene deficiency combined with myocardial infarction, filling the gap in animal models in this field.

[0037] (2) The homozygous MYL4p.E11K point mutation rat model used in this invention has a stable genotype (homozygous rate of offspring ≥95%), a high success rate of myocardial infarction model constructed by coronary artery ligation (≥80%), and a high incidence of spontaneous sustained ventricular tachycardia after myocardial infarction (≥90%), and the model has good stability and reproducibility.

[0038] (3) The spontaneous continuous ventricular tachycardia rat model constructed in this invention can be used to screen anti-ventricular tachycardia drugs (such as verifying the effect of drugs on LVEF) and develop cardioversion devices. Compared with existing models, the drug screening cycle is shortened by 30% (because the model has continuous attacks and does not require frequent induction). This model can also be used to study in depth the molecular mechanism and signaling pathway of MYL4 gene defects and myocardial infarction synergistically inducing ventricular tachycardia, and has good clinical application value.

[0039] In summary, this invention utilizes the synergistic effect of conserved gene defects (such as the MYL4 gene) and acquired myocardial infarction as dual pathological factors, achieving an induction rate ≥90% and a spontaneous duration of 1-3 minutes, significantly superior to existing methods for constructing spontaneous sustained ventricular tachycardia models. This invention can successfully induce a stable, spontaneous, sustained ventricular tachycardia pathological state in rats, and these rats can survive continuously. This model can simulate the pathophysiological process of ventricular tachycardia following myocardial infarction in humans with MYL4 gene-related cardiovascular diseases, providing an ideal experimental platform for studying the pathogenesis of arrhythmias after myocardial infarction, drug screening, and treatment development, and has significant scientific and clinical application value. Attached Figure Description

[0040] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram of the electrocardiogram / Holter monitoring results of homozygous MYL4 gene defect rats used in one embodiment of the present invention; in which, due to the MYL4E11K mutation, the atrial p wave disappears, but the QRS complex is normal and there is no ventricular arrhythmia (baseline fluctuation is electromagnetic interference). Figure 2 This is a Masson diagram of the ventricular myocardium of a homozygous MYL4 gene defective rat used in one embodiment of the present invention; Figure 3 This is a schematic diagram showing the results of continuous or intermittent long-term electrocardiogram monitoring of a certain MYL4+MI group rat 1-4 weeks after myocardial infarction in one embodiment of the present invention. Figure 4 An electrocardiogram of atrial fibrillation-ventricular tachycardia was observed in a rat in a MYL4+MI group after myocardial infarction in one embodiment of the present invention. Figure 5 In one embodiment of the present invention, a rat in a MYL4+MI group showed an electrocardiogram with frequent premature ventricular contractions and ventricular tachycardia after myocardial infarction. Figure 6 In one embodiment of the present invention, a rat in a MYL4+MI group exhibited spontaneous ventricular tachycardia after myocardial infarction. Figure 7 In one embodiment of the present invention, a rat in a certain MYL4+MI group developed spontaneous ventricular tachycardia with biphasic atrial stillness-a wave electrocardiogram after myocardial infarction; Figure 8 This is a statistical chart showing the results of surface electrocardiogram monitoring of rats in the MYL4+MI group and the WT+MI group in one embodiment of the present invention; Figure 9 This is an echocardiogram of a selected rat model of spontaneous sustained ventricular tachycardia used to test cardiac function in one embodiment of the present invention. Figure 10 Masson staining image of a pathological section of heart tissue from a selected spontaneous sustained ventricular tachycardia rat model, as shown in one embodiment of the present invention. Figure 11 This is a PCR detection result of the expression levels of relevant arrhythmia regulatory genes (such as Kcnd2, kcnj8, kcnd3, SCN5A, RYR2, ATP2A2) in a selected spontaneous sustained ventricular tachycardia rat model according to an embodiment of the present invention. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0042] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0043] Example 1 - Construction of an animal model of spontaneous sustained ventricular tachycardia This embodiment presents a preferred method for constructing an animal model of spontaneous sustained ventricular tachycardia induced by MYL4 gene deficiency combined with myocardial infarction, specifically including the following steps: (1) Constructing MYL4 gene-deficient rats, breeding and raising homozygous MYL4 gene-deficient rats (i.e., homozygous MYL4p.E11K point mutation rat model), the WT rats used were SPF grade SD rats, the steps were as follows: ① Designing sequences related to gene editing targeting wild-type MYL4: Designing primers and precursor sgRNA sequences (5'-CCTTGGCAGTCTCCTTCTG-3', 5'-AGAAGCCTGAGCCCAAGAG-3'), ssODN single-chain oligonucleotides, sgRNA scaffold sequences targeting the homologous site of human MYL4 point mutation in exon 1 of the rat MYL4 gene; ② In vitro preparation of sgRNA: PCR reaction to obtain sgRNA scaffold products; the obtained sgRNA The scaffold product was subjected to an overlap PCR reaction with the precursor sgRNA to obtain the sgRNA transcription precursor product; then the sgRNA transcription precursor product was transcribed in vitro and purified to obtain the sgRNA product; ③ In vitro preparation of Cas9 mRNA: the SP6-Cas9 plasmid was linearized using the restriction endonuclease Not I; the linearized SP6-Cas9 product was purified and transcribed to obtain the Cas9 mRNA; ④ In vitro preparation of microinjection mixture: the sgRNA and Cas9 were incubated on ice. mRNA and ssODN were mixed to prepare a microinjection mixture; ⑤ Microinjection and embryo transfer: The microinjection mixture was injected into fertilized eggs, and after culturing, the injected fertilized eggs were transferred into the oviducts of pseudopregnant female mice; ⑥ Gene identification and screening: After the pseudopregnant female mice were raised for a period of time, their offspring were born, and gene identification was performed by PCR reaction and the offspring with positive genotypes were screened; the offspring with positive genotypes were mated with wild-type rats, and after their offspring were born, gene identification and screening were performed to finally obtain a homozygous MYL4p.E11K point mutant rat model; Regarding the pathological characteristics (no spontaneous sustained ventricular tachycardia) of the homozygous MYL4 gene-deficient rats used: MYL4 mainly affects the atria and is also expressed in Purkinje fibers. Under normal physiological conditions, no ventricular arrhythmias occur. Specifically: 1) Changes are mainly in atrial structure and function, such as weakened atrial dilatation / contraction, but do not directly lead to ventricular arrhythmias. 2) Ventricular structure and electrophysiology are relatively stable, with no significant ventricular hypertrophy, fibrosis, or conduction / repolarization abnormalities. 3) Electrocardiogram / Holter monitoring: results are as follows... Figure 1As shown, QRS duration and QT interval after myocardial infarction showed no statistically significant differences between MYL4-deficient homozygous rats and wild-type rats; and MYL4-deficient homozygous rats without modeling did not exhibit spontaneous premature ventricular contractions, non-sustained or sustained ventricular tachycardia. 4) Ventricular myocardial pathology: No extensive necrosis or fibrosis, insufficient to form the ventricular tachycardia matrix (Masson diagram as shown). Figure 2 (as shown); 5) Under physiological conditions, this model does not have the pathological basis for spontaneous sustained ventricular tachycardia, and is an ideal "first-hit" model for studying malignant ventricular arrhythmias induced by gene defects combined with secondary blows (such as myocardial ischemia).

[0044] (2) Construction of the myocardial infarction model: Healthy homozygous MYL4p.E11K point mutant rats aged 8-10 weeks and weighing 250-300 g were selected and acclimatized for 1 week before surgery. The rats were anesthetized by intraperitoneal injection of 2.5% tribromoethanol (6 mL / kg) (the latest anesthetic, which meets ethical requirements), and fixed supine on the operating table. A midline incision was made in the neck, the trachea was separated and an endotracheal tube was inserted, and the rats were connected to a small animal ventilator (respiratory rate 80 breaths / min, tidal volume 10 mL / kg). An incision was made in the 4th-5th intercostal space on the left side of the chest, and the pectoralis major and pectoralis minor muscles were separated layer by layer. The thoracic cavity was opened with a rib retractor to expose the heart. Under the surgical microscope, the left anterior descending coronary artery was located between the left atrial appendage and the pulmonary conus. The left coronary artery was ligated with 6-0 non-traumatic sutures about 2 mm from the main trunk of the left coronary artery. After ligation, the color of the anterior wall of the left ventricle changed from red to pale, and the myocardial motility was significantly weakened, indicating that the myocardial infarction model was successfully constructed. The thoracic cavity was closed layer by layer, expelling air during the closure process. The endotracheal tube was removed after the rat regained spontaneous breathing. Postoperatively, the rat was placed in a warm environment for resuscitation and given intraperitoneal injections of penicillin (200,000 U / kg) once daily for 3 consecutive days to prevent infection.

[0045] (3) After the myocardial infarction model is established, long-term electrocardiogram monitoring (continuous or intermittent) is performed on the animals for 1-4 weeks. Animals with spontaneous continuous ventricular tachycardia (3 or more consecutive premature ventricular contractions with a duration of ≥30s) are selected as the target rat model. The specific screening criteria are as follows: 1-4 weeks after surgery, under anesthesia (each time isoflurane-induced anesthesia), monitoring for 3 minutes, the occurrence of ≥3 consecutive premature ventricular contractions with a duration of ≥30s is judged as spontaneous continuous ventricular tachycardia.

[0046] (4) After the target rat model is selected, the target rat model can be validated to confirm the accuracy and reliability of the above screening criteria. The validation analysis includes: cardiac function test: left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDD), and left ventricular end-systolic diameter (LVESD); pathological section observation: heart tissue is taken to make pathological sections and HE staining and Masson staining are performed; gene expression detection: real-time fluorescence quantitative PCR technology is used to detect the expression level of MYL4 gene and / or related arrhythmia regulatory genes (such as KCNQ1, SCN5A, CACNA1C) in the myocardial tissue of model rats.

[0047] Example 2 - Screening of a rat model of spontaneous sustained ventricular tachycardia This embodiment describes a preferred screening procedure for a rat model of spontaneous sustained ventricular tachycardia. After the myocardial infarction model is constructed as described in step (2) of Embodiment 1, surface electrocardiogram monitoring and further verification analysis are performed on the rats, as detailed below: (1) A total of 60 rats were used in the experiment. 30 rats were the MYL4 gene defect combined with myocardial infarction rat model constructed by the method in Example 1 (MYL4+MI group), and 30 rats were WT rats (SPF grade SD strain rats) combined with myocardial infarction rat model (WT+MI group). (2) Surface ECG monitoring: After rats were anesthetized with isoflurane, they were fixed to a table, and the electrode leads of the LabChart ECG monitoring system were fixed to the limbs of the rats through subcutaneous punctures. Surface ECGs were recorded. Continuous or intermittent long-term ECG monitoring was performed on rats 1-4 weeks after myocardial infarction. The monitoring results of one rat model are as follows: Figure 3 As shown, based on the electrocardiogram results, rats exhibiting spontaneous sustained ventricular tachycardia (defined as three or more consecutive premature ventricular contractions lasting ≥30 seconds) were selected, which became the animal model of spontaneous sustained ventricular tachycardia induced by MYL4 gene deficiency combined with myocardial infarction.

[0048] The surface electrocardiograms of some MYL4+MI group rats in the above experimental rats are shown in Figure 4~. Figure 7 As shown in Table 1, the statistical results of surface electrocardiogram monitoring of all rats are presented in Table 2 and 3. Figure 8 As shown.

[0049] The monitoring results above show that: in the MYL4-MI group, one animal died of ventricular fibrillation during recording and was not included in the statistics; in the WT+MI group: 16 animals had no ventricular arrhythmia, 7 animals had single premature ventricular contractions (PVCs), 4 animals had PVCs in bigeminy, 2 animals had short runs of ventricular tachycardia, 1 animal had medium-range ventricular tachycardia, 0 animals had long-range ventricular tachycardia, 0 animals had torsades de pointes, and 0 animals had ventricular fibrillation; in the MYL4+MI group: 2 animals had no ventricular arrhythmia, 1 animal had single premature ventricular contractions (PVCs), 1 animal had PVCs in bigeminy, 2 animals had short runs of ventricular tachycardia, 4 animals had medium-range ventricular tachycardia, 13 animals had long-range ventricular tachycardia, 5 animals had torsades de pointes, and 1 animal had ventricular fibrillation.

[0050] The selected model rats underwent long-term electrocardiogram monitoring (for 4 weeks), (e.g.) Figure 3 The results (as shown) indicated that the model rats experienced multiple spontaneous sustained ventricular tachycardia episodes per week, with each episode lasting 1-3 minutes, suggesting that the model has stable spontaneous sustained ventricular tachycardia episode characteristics.

[0051] The above results indicate that, compared with the simple myocardial infarction model (induction rate of 20%-30%), the induction rate of ventricular tachycardia in this model is ≥90%, of which ventricular tachycardia accounts for 80%, and it can spontaneously and continuously occur (1~3 min) without electrical stimulation. The success rate of constructing a rat model of spontaneous continuous ventricular tachycardia is ≥80%.

[0052] (3) In order to further verify the accuracy and reliability of the above screening criteria, this embodiment also performs the following verification analysis on the spontaneous sustained ventricular tachycardia animal model obtained through screening: 1) Cardiac function testing; A rat exhibiting spontaneous sustained ventricular tachycardia, selected from those screened by surface electrocardiogram monitoring as described in (2) above, was subjected to cardiac function testing. Specifically, this included: using echocardiography to assess the cardiac function of the model rat, such as... Figure 9 As shown, the results indicated that the left ventricular ejection fraction (LVEF) of the model rats was significantly reduced (<40%), while the left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were significantly increased, indicating that the model rats had obvious cardiac dysfunction, which was consistent with the changes in cardiac function in human patients with MYL4 gene deficiency and myocardial infarction.

[0053] 2) Observation of pathological sections; A rat exhibiting spontaneous sustained ventricular tachycardia, selected from the above (2) surface electrocardiogram monitoring, was subjected to pathological section observation. The specific steps included: after euthanizing the model rat, heart tissue was taken to prepare pathological sections and Masson staining was performed. The steps are briefly described below: ① Dewaxed paraffin sections were sequentially rinsed with xylene (I, II) for 5 min each, then with anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 3 min each, and then rinsed with distilled water for 2 min.

[0054] ② Hematoxylin staining: Immerse in hematoxylin staining solution for 5-10 min → rinse with tap water, differentiate with differentiation solution for 30 s → return to blue with tap water for 10 min → rinse with distilled water for 1 min.

[0055] ③ Stain with Masson's compound dye solution, then stain with Ponceau S-Acid Fuchsin solution for 5-10 minutes → rinse quickly with distilled water.

[0056] ④ Differentiation and fixation: Treat with 1% phosphomolybdic acid solution for 3-5 min → directly stain with aniline blue solution for 5-10 min (no need to wash with water).

[0057] ⑤ Dehydrate and clear the slide, wash with 1% glacial acetic acid solution for 1 min → dehydrate with anhydrous ethanol twice for 3 min → clear with xylene twice for 5 min → seal with neutral resin.

[0058] ⑥ Result interpretation: Collagen fibers appear blue, muscle fibers and cytoplasm appear red, and cell nuclei appear blue-purple.

[0059] Masson staining results are as follows: Figure 10 As shown, the model rats exhibit a large amount of collagen fiber deposition in the myocardial interstitium, indicating that the model rats have obvious myocardial damage and fibrosis changes, which are consistent with the pathological changes of the heart after myocardial infarction.

[0060] 3) Gene expression detection; Gene expression detection was performed on a rat that exhibited spontaneous sustained ventricular tachycardia, selected from the above (2) surface electrocardiogram monitoring screening. Specifically, the expression levels of relevant arrhythmia regulatory genes (such as Kcnd2, kcnj8, kcnd3, SCN5A, RYR2, and ATP2A2) in the myocardial tissue of the model rat were detected using real-time quantitative PCR technology. The detection steps are briefly described below: ① Nucleic acid template preparation: Extract the target nucleic acid (DNA or RNA) from the sample to be tested; if the target nucleic acid is RNA, it needs to be synthesized into cDNA through reverse transcription reaction as a template for qPCR amplification, ensuring that the purity and integrity of the template nucleic acid meet the amplification requirements.

[0061] ② Reaction system preparation: Under ice bath conditions, the fluorescent premixed solution, specific primers, nucleic acid template and enzyme-free water were mixed in a preset ratio to construct the qPCR reaction system; negative control (without template), positive control and internal reference gene control were set up simultaneously to verify the effectiveness of the reaction and correct errors.

[0062] ③ Amplification on the instrument: Place the prepared reaction system in the qPCR instrument, set the amplification program (including pre-denaturation, denaturation-annealing-extension cycle, and melting curve analysis stage), start the amplification reaction, and collect fluorescence signals in real time.

[0063] ④ Results Analysis: Based on the fluorescence signal data collected by the qPCR instrument, the relative or absolute expression level of the target gene was determined by calculating the Ct value and correcting the internal reference gene, thus completing the detection.

[0064] PCR test results as follows Figure 11 As shown, the results also showed significant abnormalities in the expression levels of Kcnd2, kcnj8, kcnd3, SCN5A, RYR2, and ATP2A2 genes in the cardiac conduction tissue of the model rats, indicating that MYL4 gene defects can affect the expression of ion channel-related genes in cardiomyocytes, thereby leading to arrhythmia.

[0065] As can be seen from the above embodiments, this invention has for the first time constructed a stable animal model of sustained ventricular tachycardia with a very high induction rate. It can simulate the pathophysiological process of ventricular tachycardia after myocardial infarction in human MYL4 gene-related cardiovascular diseases. It provides an ideal experimental vehicle for studying the pathogenesis of arrhythmias after myocardial infarction, drug screening and treatment development, and has important scientific research and clinical application value.

[0066] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A method for constructing an animal model of cardiac arrhythmia, characterized in that, Including the following steps: Step A: Select conserved arrhythmia-related genes based on the target species, and construct and select homozygous animal models with gene defects; Step B: Construct an animal model of myocardial injury using the homozygous gene defect animal model obtained in Step A; Step C: Screen animal models of cardiac arrhythmia that meet the criteria through phenotypic monitoring; The arrhythmia animal models include atrial fibrillation arrhythmia animal models, ventricular fibrillation arrhythmia animal models, spontaneous sustained ventricular tachycardia animal models, bradycardia animal models, and atrioventricular reentrant tachycardia animal models.

2. The construction method according to claim 1, characterized in that, The conserved arrhythmia-related genes include SCN5A, KCNQ1, KCNH2, CACNA1C, GJA1, RYR2, CASQ2, MYL4; and / or, The animal model of myocardial injury is constructed by one of the following methods: permanent coronary artery ligation model construction, drug / chemical induction model construction, electrical stimulation induction model construction, ischemia / reperfusion injury model construction, physical / mechanical injury model construction, and genetic engineering model construction.

3. The construction method according to claim 1, characterized in that, The steps for constructing the spontaneous sustained ventricular tachycardia animal model include: S1. Construct animal models of MYL4 gene defect, breed and raise animals with homozygous MYL4 gene defect, and select animal models of MYL4 gene defect. S2. Using the MYL4 gene defect homozygous animal model obtained in step S1, a myocardial infarction animal model was constructed by ligation of the left anterior descending coronary artery. S3. After constructing the animal model of myocardial infarction as described in step S2, long-term electrocardiogram monitoring is performed on the animals, and the animal model of spontaneous sustained ventricular tachycardia induced by MYL4 gene deficiency combined with myocardial infarction is screened based on the monitoring results.

4. The construction method according to claim 3, characterized in that, The animals mentioned include rodents, lagomorphs, canines, and suidae.

5. The construction method according to claim 3, characterized in that, The animal is a rat; in step S1, the MYL4 gene defective animal model is a MYL4 gene defective rat model, which includes MYL4 gene knockout rats and MYL4 functional defective rats caused by gene editing.

6. The construction method according to claim 5, characterized in that, In step S1, the MYL4 gene defect homozygous rat model is a homozygous MYL4p.E11K point mutation rat model; and / or, In step S1, the MYL4 gene expression level in the MYL4 gene-deficient homozygous rat model decreased by more than 90% compared with the wild type, meeting the conditions for inducing ventricular tachycardia after myocardial infarction.

7. The construction method according to claim 5, characterized in that, In step S2, the steps for constructing a rat model of myocardial infarction include: after anesthetizing the rat, it is fixed supine on the operating table, a midline incision is made in the neck, the trachea is separated and intubated, and a small animal ventilator is connected; an incision is made in the 4th-5th intercostal space on the left side of the chest, the muscles are separated layer by layer, the thoracic cavity is opened, and the heart is exposed; the left anterior descending coronary artery is located between the left atrial appendage and the pulmonary conus, and ligation is performed near the main trunk of the left coronary artery using atraumatic cotton suture; successful ligation is indicated by the observation that the anterior wall of the left ventricle turns pale and myocardial movement weakens; the thoracic cavity is closed layer by layer, and the tracheal tube is removed after the rat's spontaneous breathing recovers. Postoperative infection prevention is necessary.

8. The construction method according to claim 5, characterized in that, In step S3, the screening criteria are as follows: 1-4 weeks post-surgery, under anesthesia, monitoring for 3 minutes, ≥3 consecutive premature ventricular contractions lasting ≥30 seconds are defined as spontaneous sustained ventricular tachycardia; and / or, The construction method further includes step S4, which involves validating and analyzing the obtained spontaneous sustained ventricular tachycardia animal model, including functional validation and / or pathological validation. The functional validation includes echocardiography to detect left ventricular ejection fraction, left ventricular end-diastolic diameter, and left ventricular end-systolic diameter. The pathological validation includes HE staining to observe myocardial necrosis and Masson staining to detect fibrosis.

9. The construction method according to claim 5, characterized in that, The success rate of establishing a spontaneous sustained ventricular tachycardia rat model is ≥80%, and the arrhythmia induction rate is ≥90%, of which ventricular tachycardia accounts for 80%.

10. An application of the construction method as described in any one of claims 1 to 9, or of the animal model of cardiac arrhythmia obtained therefrom, characterized in that, The application is selected from at least one of the following applications: the application of animal models of arrhythmia in determining the molecular mechanism by which gene defects and myocardial injury synergistically induce arrhythmia; the application of animal models of arrhythmia in screening antiarrhythmic drugs; and the application of animal models of arrhythmia in the preparation of medical devices for the treatment of arrhythmia.

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