Use of myzap overexpression recombinant vector for preparing a drug for preventing and treating arrhythmia

Abstract

The application relates to application of a MYZAP overexpression recombinant vector in preparation of a drug for preventing and treating arrhythmia, and belongs to the technical field of biological medicines. In order to solve the problem that the application of a gene drug is limited in prevention and treatment of arrhythmia after myocardial infarction, the application provides application of a myocardial zonula adherens protein MYZAP overexpression recombinant vector in preparation of a drug for preventing and treating arrhythmia, wherein the MYZAP overexpression recombinant vector is an adeno-associated virus vector AAV9-MYZAP overexpressing a MYZAP gene. In-vivo experiments prove that MYZAP overexpression can increase the MYZAP protein content in cardiac muscle cells of a heart, reduce the occurrence of arrhythmia after myocardial infarction, and improve the cardiac function and cardiac electrical conduction disturbance after myocardial infarction. Based on this, the application further provides a drug for preventing and treating arrhythmia after myocardial infarction, and provides a new strategy for gene therapy of arrhythmia after myocardial infarction, and has a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of a MYZAP overexpression recombinant vector in preparation of a drug for preventing and treating arrhythmia, in particular to application in preparation of a drug for preventing and treating post-myocardial infarction arrhythmia. BACKGROUND

[0002] Myocardial infarction (MI) refers to myocardial necrosis caused by acute or persistent myocardial ischemia or hypoxia. After MI, macrophages mediate the release of various pro-inflammatory mediators and activate immune signaling pathways, playing an important role in inflammation, leading to electrical remodeling and structural remodeling of myocardial cells, thereby promoting the occurrence and development of arrhythmia.

[0003] Existing drugs for treating post-myocardial infarction arrhythmia include (1) calcium channel blockers, such as verapamil and diltiazem. Such drugs have the effect of relaxing vascular smooth muscle and reducing vascular tension; (2) beta blockers, such as propranolol. Such drugs can slow the heart rate and inhibit myocardial contractility; (3) sodium channel blockers, such as lidocaine, which can block the activation state and inactivation state of the sodium channel, increase the excitation threshold, and reduce automaticity; (4) drugs that prolong the action potential duration, such as amiodarone, which can prolong the action potential and effective refractory period of myocardial tissue in various parts.

[0004] Although these drugs have certain efficacy in treating post-myocardial infarction arrhythmia, they also have some side effects. Some anti-arrhythmic drugs can inhibit the normal contraction function of the heart, leading to a decrease in cardiac output and, in severe cases, heart failure. Some drugs can dilate blood vessels, leading to a decrease in blood pressure and symptoms such as dizziness and fatigue. Some drugs can cause irregular heart rate, including tachycardia or bradycardia, causing symptoms such as palpitations and chest tightness.

[0005] Gene drug therapy can precisely target diseased genes and intervene at specific molecular targets, and its mechanism is clear, which helps to reduce potential side effects. However, gene drugs have not been widely used in the prevention and treatment of post-myocardial infarction arrhythmia. The main reason is that the pathophysiological mechanism involves complex regulation of multiple genes and signaling pathways, making it difficult to identify and verify effective treatment targets, thereby limiting the precise selection of gene targets. SUMMARY

[0006] To solve the problem of limited application of gene drugs in the prevention and treatment of post-myocardial infarction arrhythmia, the present application provides the application of MYZAP as a multi-ion channel target in the preparation of a drug for preventing and treating post-myocardial infarction arrhythmia.

[0007] Technical scheme of the present application:

[0008] The application provides an application of a myocardial zonula adherens protein (MYZAP) overexpression recombinant vector in preparation of a drug for preventing and treating arrhythmia, and the arrhythmia is post-myocardial infarction arrhythmia.

[0009] Further, the MYZAP overexpression recombinant vector is an adeno-associated virus vector AAV9-MYZAP overexpressing the MYZAP gene.

[0010] Further, the nucleotide sequence of the MYZAP gene is shown in SEQ ID NO. 1.

[0011] Further, the drug for preventing and treating arrhythmia can reduce the occurrence of post-myocardial infarction arrhythmia, and improve post-myocardial infarction cardiac function and cardiac electrical conduction disorder by increasing the MYZAP protein content in cardiac muscle cells.

[0012] A drug for preventing and treating post-myocardial infarction arrhythmia, the drug contains a MYZAP gene overexpression recombinant vector, and the nucleotide sequence of the MYZAP gene is shown in SEQ ID NO. 1.

[0013] Further, the MYZAP gene overexpression recombinant vector is an adeno-associated virus vector AAV9-MYZAP overexpressing the MYZAP gene.

[0014] The application has the following beneficial effects:

[0015] The application takes the myocardial zonula adherens protein MYZAP gene as a drug target, constructs an AAV9-mediated MYZAP gene overexpression recombinant vector and a mouse myocardial infarction model, and proves through in-vivo experiments that MYZAP overexpression can reduce the occurrence of post-myocardial infarction arrhythmia, and improve post-myocardial infarction cardiac function and cardiac electrical conduction disorder. Based on this, the application provides an application of the MYZAP gene as a drug target in preparation of a drug for preventing and treating post-myocardial infarction arrhythmia, provides a new strategy for gene therapy of post-myocardial infarction arrhythmia, and has a wide clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a volcano plot of single-cell RNA sequencing of myocardial cells of two groups of mice in Example 1;

[0017] Figure 2 It is a WB result graph of MYZAP expression levels of myocardial cells of two groups of mice in Example 1;

[0018] Figure 3 It is an image of MYZAP immunofluorescence staining of myocardial cells of two groups of mice in Example 1;

[0019] Figure 4 Fluorescence intensity comparison chart of MYZAP immunofluorescence staining of myocardial cells in two groups of mice in Example 1;

[0020] Figure 5 Echocardiogram of each group of mice in Example 2;

[0021] Figure 6 Comparison chart of ejection fraction EF and left ventricular fractional shortening FS of each group of mice in Example 2;

[0022] Figure 7 Comparison chart of left ventricular end-diastolic diameter LVIDd and end-systolic diameter LVIDs of each group of mice in Example 2;

[0023] Figure 8 Comparison chart of left ventricular posterior wall end-diastolic diameter LVPWd and end-systolic thickness LVPWs of each group of mice in Example 2;

[0024] Figure 9 Electrocardiogram of each group of mice in Example 3;

[0025] Figure 10 Comparison chart of ventricular arrhythmia induction time of each group of mice in Example 3;

[0026] Figure 11 Comparison chart of ventricular arrhythmia incidence of each group of mice in Example 3;

[0027] Figure 12 Fluorescence photos of heart staining of each group of mice in Example 4;

[0028] Figure 13 Comparison chart of heart conduction velocity of each group of mice in Example 4;

[0029] Figure 14 Comparison chart of action potential time APD of each group of mice in Example 4;

[0030] Figure 15 Comparison chart of action potential AP rising speed of each group of mice in Example 4;

[0031] Figure 16 Comparison chart of action potential AP peak value of each group of mice in Example 4;

[0032] Figure 17 Ventricular action potential APD 50 and APD 30 representative cardiac surface map;

[0033] Figure 18 Ventricular action potential APD 50 and APD 30Comparison chart;

[0034] Figure 19 Echocardiograms of mice in each group in Example 5;

[0035] Figure 20 This is a comparison chart of ejection fraction (EF) and left ventricular fractional shortening (FS) of mice in each group in Example 5.

[0036] Figure 21 This is a comparison of the left ventricular end-diastolic LVIDd and end-systolic LVIDs of mice in each group in Example 5.

[0037] Figure 22 This is a comparison of the end-diastolic left ventricular posterior wall thickness (LVPWd) and end-systolic left ventricular thickness (LVPWs) in each group of mice in Example 5.

[0038] Figure 23 Electrocardiograms of mice in each group in Example 6;

[0039] Figure 24 This is a comparison of the induction time of ventricular arrhythmias in each group of mice in Example 6;

[0040] Figure 25 This is a comparison chart of the incidence of ventricular arrhythmias in each group of mice in Example 6;

[0041] Figure 26 These are fluorescent photographs of the mice stained in each group in Example 7;

[0042] Figure 27 This is a comparison chart of cardiac conduction velocities in different groups of mice in Example 7;

[0043] Figure 28 This is a comparison chart of action potential duration (APD) of mice in each group in Example 7;

[0044] Figure 29 This is a comparison chart of the rise rate of action potentials (AP) in each group of mice in Example 7;

[0045] Figure 30 This is a comparison chart of the peak action potentials (APs) of mice in each group in Example 7;

[0046] Figure 31 The action potentials (APDs) of the ventricles in each group of mice in Example 7. 50 and APD 30 Representative cardiac surface atlas;

[0047] Figure 32 For each group of mice in Example 7, ventricular APD 50 and APD 30 The comparison chart. Detailed Implementation

[0048] The technical solutions of the present application are further illustrated below in connection with the examples, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present application without departing from the spirit and scope of the present application shall be encompassed in the protection scope of the present application. The process equipment or device not specifically mentioned in the following examples is the conventional equipment or device in the art. If not specifically mentioned, the raw materials used in the examples of the present application are commercially available. If not specifically mentioned, the technical means used in the examples of the present application are conventional means known to those skilled in the art.

[0049] Example 1

[0050] This example demonstrates that the expression of MYZAP gene in the marginal tissue of myocardial infarction in mice is reduced.

[0051] To elucidate the role of MYZAP in myocardial infarction injury, this example first constructs a mouse myocardial infarction model, and the specific construction method is as follows:

[0052] Male C57BL / 6 mice weighing about 25 g were purchased and adaptively fed, and then a MI model was constructed by ligating the left anterior descending branch of the mouse heart. After weighing the mice, they were anesthetized by intraperitoneal injection of anesthetic Avertin (0.22 g / kg, Sigma, St. Louis, MO, USA). The mice were fixed in a supine position on the operating table, and the skin was shaved. A respirator was inserted to prevent pneumothorax, and then the muscles between the 2nd and 3rd ribs on the left chest were bluntly separated to expose the heart. The left anterior descending branch of the heart was ligated with 7 / 0 medical suture, and the apex of the heart turned white after ligation. Then the electrocardiogram was detected, and the appearance of ST segment elevation in electrocardiogram indicated that the model was successfully established. Then the left chest skin was sutured, and the mouse was placed on a 37°C heating pad. After complete recovery and good condition, the mouse was placed in a warm environment and continued to be fed for 12 hours. The sham operation group was treated in the same way as the model group except that it was not ligated.

[0053] (1) Single cell RNA sequencing

[0054] The myocardial cells of the MI group mice and the myocardial cells of the Sham group mice were taken for single cell RNA sequencing, and the sequencing results are shown in the volcano plot. Figure 1 After myocardial infarction, 455 genes were up-regulated and 477 genes were down-regulated, among which the expression of MYZAP gene was significantly down-regulated.

[0055] (2) Western blot experiment

[0056] Western blot experiment was performed on myocardial cells at the edge of myocardial infarction of the mice in the MI group and myocardial cells at the same position of the mice in the Sham group. 150 μl of cell lysate prepared in advance was added to the extracted cells (the amount of lysate added can be adjusted according to the cell density), and the cells were lysed in an ice bath for 5 minutes. The adherent cells were scraped off with a cell scraper, and the protein lysate was collected into a 1.5 ml EP tube with a pipette. Then, a centrifuge was used at 13500 rpm and 4°C for 15-20 minutes. After centrifugation, the supernatant was aspirated into a new 1.5 ml EP tube with a pipette. The supernatant was the total protein extracted. First, the prepared gel was placed in the electrophoresis tank, and the electrophoresis solution was added to the appropriate scale. The protein samples were loaded in order according to the experimental requirements, and then the protein marker was added. The electrophoresis device was connected, and the lamination gel was adjusted to a constant voltage of 70 V. The electrophoresis was run for about 30 minutes. When the protein sample passed through the lamination gel and formed a straight line, the voltage was adjusted to 110 V. The gel was taken out with a gel plate, and was placed in the order of white sponge-filter paper-NC membrane-gel-filter paper-black sponge. The bubbles were gently chased away. The membrane transfer tank was placed in ice to keep it cool, and the constant current was 300 mA. The membrane transfer was performed for 120 minutes. The NC membrane was placed in the blocking solution containing 10% skim milk prepared in advance, and was blocked in a shaking bed at room temperature for 1.5 hours. According to the molecular weight of the target protein, the blocked NC membrane was cut into the desired band, and was placed in the one-antibody dilution solution diluted in proportion in advance. The clamped chain bag was incubated overnight at 4°C. The NC membrane was taken out and washed with PBST for 7 minutes each time for a total of four times. Then, the diluted secondary antibody solution was incubated at room temperature for 50 minutes, and then developed and analyzed.

[0057] The results are shown in Figure 2 Compared with the Sham group, the expression of MYZAP gene in the myocardial cells of the MI group was significantly reduced.

[0058] (3) Immunofluorescence

[0059] Immunofluorescence staining was performed on myocardial cells of the MI group and the Sham group. The myocardial cells were inoculated in a 24-well plate coated with a glass slide at a density of 50% and placed in a cell culture box for 24 hours. The cell culture medium was discarded with a pipette, and the cells were washed with PBS for 5 minutes at a time for a total of 3 times. The cells were fixed with pre-cooled 4% paraformaldehyde for 20 minutes. Prepare the penetration solution: 30 μl Triton in 10 ml PBS. Add an appropriate amount of penetration solution to each well to cover the cell fragments, and let stand at room temperature for 1 hour. Block the cells with goat serum at 37°C for 1 hour. After blocking, discard the blocking solution and wash with PBST for 5 minutes at a time for a total of 3 times. Dilute the primary antibody according to the instructions and place it in a 4°C refrigerator overnight. Wash the primary antibody with PBST for 5 minutes at a time for a total of 3 times. Prepare the fluorescent secondary antibody according to the proportion, and add 50 μl of prepared fluorescent secondary antibody to each well in the dark. Incubate at 37°C for 1.5 hours, and wash the secondary antibody with PBST for 5 minutes at a time for a total of 3 times. Prepare DAPI according to the proportion in the dark, and add 50 μl of prepared DAPI to each well. Let stand at room temperature for 15 minutes, discard the DAPI staining solution, and wash the cells with PBST for 5 minutes at a time for a total of 3 times. Place the treated cells in a laser confocal microscope under dark conditions for imaging.

[0060] The results are shown in Figure 3 and Figure 4 Compared with the Sham group, the expression of the MYZAP gene in the myocardial cells of the MI group was significantly reduced, confirming that the MYZAP gene is down-regulated in myocardial cells after myocardial infarction.

[0061] Example 2

[0062] In this example, a recombinant vector for overexpression of the MYZAP gene, an adeno-associated virus vector AAV9-MYZAP, was constructed, and it was confirmed through in vivo experiments that overexpression of the MYZAP gene could significantly improve the cardiac function of mice with myocardial infarction.

[0063] Adeno-associated virus (AAV) is an effective and safe transduction vector, and different serotypes have different tissue infectivity and gene delivery efficiency. In this example, the AAV9 serotype with the strongest infection efficiency in myocardial tissue was selected.

[0064] MYZAP gene overexpression plasmid construction and adeno-associated virus packaging were completed by Guangzhou Aizhe Biotechnology Co., Ltd.: using AAV9 plasmid as a carrier, inserting the MYZAP full-length sequence as shown in SEQ ID NO. 1 and the enhanced green fluorescent protein (EGFP) sequence, and using CMV as a universal promoter for various types of cells. After successful construction of the MYZAP gene overexpression recombinant vector, it was stored at -80℃ for standby. All virus-coated plasmids were verified by sequencing.

[0065] To verify the effect of MYZAP overexpression on cardiac function in myocardial infarction mice, male C57BL / 6 mice (about 25 g) were injected with MYZAP gene overexpression recombinant vector AAV9-MYZAP via the tail vein, with an injection dose of 2×10 11 GC (genome containing particles, genome-containing particles), with normal saline (150 μl per mouse) as the solvent. The control group of mice was also injected with the same dose of adeno-associated virus carrying a negative control sequence (+NC) via the tail vein.

[0066] After 4 weeks of injection, a myocardial infarction model was established, and the mice were divided into Sham sham operation group, MI model group, +MYZAP overexpression group and +NC negative control group. Echocardiography was performed on each group of mice to detect the cardiac function of the mice.

[0067] The method of cardiac ultrasound detection is as follows:

[0068] After weighing the mice, they were anesthetized by intraperitoneal injection of anesthetic Averdin. They were fixed in a supine position on a 37℃ constant temperature operation platform, and their limbs were fixed with tape. After being shaved, coupling agent was applied to the front of the mouse's chest. The left ventricular long-axis and left ventricular short-axis papillary muscle level M-Mode curve was measured, and the instrument analysis software was used to determine the indicators of the mouse heart. The VINNO 6 high-resolution imaging system was used for analysis. The detection indicators included left ventricular end-diastolic and end-systolic diameter (LVIDd / LVIDs), left ventricular posterior wall end-diastolic and end-systolic thickness (LVPWd / LVPWs), ejection fraction (EF%) and left ventricular fractional shortening (FS%) to assess the cardiac function of the mice.

[0069] The results are as follows:Figures 5-8 As shown, compared with the Sham group, the left ventricular ejection fraction (EF%) and left ventricular fractional shortening (FS%) of the myocardial infarction mice were significantly reduced, the left ventricular internal diameter at end diastole (LVIDd), left ventricular internal diameter at end systole (LVIDs) were significantly increased, and the left ventricular posterior wall thickness at end diastole (LVPWd), left ventricular posterior wall thickness at end systole (LVPWs) were significantly reduced. The above data showed that the myocardial infarction mice had obvious heart dysfunction. However, it is worth noting that compared with the NC group, overexpression of MYZAP improved the heart function indicators EF%, FS%, LVIDd, LVIDs and LVPWd of the myocardial infarction mice. The negative control group did not affect the heart dysfunction caused by myocardial infarction.

[0070] Example 3

[0071] In this embodiment, the MYZAP gene overexpression recombinant vector, adeno-associated virus vector AAV9-MYZAP, was constructed, and it was verified by in vivo experiment that overexpression of MYZAP gene could significantly reduce the occurrence of arrhythmia in myocardial infarction mice.

[0072] In this embodiment, based on the mouse models in different groups in Example 3, the mice in each group were subjected to programmed electrical stimulation, and the incidence of ventricular arrhythmia after MI was detected.

[0073] The specific method of programmed electrical stimulation is as follows:

[0074] After the mice were anesthetized, they were fixed on their backs under a stereomicroscope. The skin was cut along the right side of the neck, the glands were bluntly separated, the jugular vein was exposed, and a small opening was cut along the right branch near the head side of the blood vessel. The electrode was inserted into the mouse right ventricle from the opening. Using a stimulation mode of 10 consecutive electrical pulses (S1) with a coupling interval of 80 ms, then applying two additional stimuli (S2 and S3) of 80, 78 ms, respectively, with a coupling interval of 2 ms, i.e. with a step of -2, the occurrence of arrhythmia was recorded at three voltages of 3.5 V, 5 V and 8 V, respectively. Adjust the waveform and start the stimulation program. Thus, the occurrence of ventricular arrhythmia was induced. The stimulation was repeated twice to check the reproducibility of the experiment.

[0075] As shown in the results, Figures 9-11 Compared with the Sham group, the incidence of ventricular arrhythmia in the MI mouse model was significantly increased, and the ventricular arrhythmia induction time was significantly prolonged. In this embodiment, it was observed that overexpression of MYZAP significantly reduced the incidence of arrhythmia induced by programmed electrical stimulation and the duration of arrhythmia in myocardial infarction mice.

[0076] Example 4

[0077] The present embodiment constructs a MYZAP gene overexpression recombinant vector, an adeno-associated virus vector AAV9-MYZAP, and proves that overexpression of MYZAP gene can improve cardiac electrical conduction disorder after myocardial infarction through in vivo experiments.

[0078] Impaired myocardial action potential conduction can lead to arrhythmia by forming a slow conduction reentry circuit. Based on the mouse model of different groups in Example 3, the present embodiment applies Optical mapping technology to detect the changes of cardiac conduction velocity and APD (Action Potential Duration) of mice in each group.

[0079] The specific method of Optical mapping is as follows:

[0080] After the mice are anesthetized and sacrificed, the thoracic cavity is quickly opened, and the heart is immediately taken out and placed in the perfusion fluid. The aorta of the heart is hung on the perfusion needle, and perfusion fluid is injected to try to remove the blood in the heart. Under the condition of 37°C, the perfusion needle is connected with the Langendorff perfusion device. After the drop speed is stable and the heart is free of blood, a decoupling agent, (-) Blebbistatin, is added to the perfusion tube to a final concentration of 20 µmol / L to stop the beating of the heart. Then, voltage-sensitive dye RH237 dye (#S1109, invitrogen) working solution is added to dye the perfused heart. After the heart is evenly dyed, MiCAM05 CMOS imaging system is used to collect the picture information of the heart and record the overall fluorescence changes of the heart.

[0081] The results are shown in Figures 12-18 Compared with the Sham group, the conduction time of the heart of the MI group mice is prolonged after passing through the same distance, which indicates that the cardiac conduction velocity of the MI group mice is significantly slowed down. Since the myocardial conduction velocity is closely related to the maximum rate of AP depolarization, the maximum rate of AP depolarization is evaluated in the present embodiment. Compared with the Sham group, the upstroke velocity of the ventricular action potential of the MI group mice is significantly reduced, and the ventricular APD 30 and APD 50 of the MI group mice are significantly prolonged. The APD 30 and APD 50 are prolonged, the action potential duration of the myocardial cells is lengthened, and the time for the cells to recover to the excitable state is delayed, which will slow down the conduction of excitation between myocardial cells and increase the risk of cardiac conduction block. As shown in the data above, the cardiac electrical conduction of the mice is impaired after MI. The present embodiment further studies and finds that overexpression of MYZAP in the mouse heart significantly increases the conduction velocity of the heart after MI, and the upstroke velocity and peak value of the action potential are also significantly increased. At the same time, overexpression of MYZAP shortens the ventricular APD 30and APD 50 .

[0082] Example 5

[0083] In this embodiment, a recombinant vector for knocking down MYZAP gene expression, an adeno-associated virus vector AAV9-Sh-MYZAP, was constructed, and it was verified through in vivo experiments that knocking down MYZAP gene can significantly reduce the heart function of mice.

[0084] The recombinant vector for knocking down MYZAP gene expression was completed by Guangzhou Aijie Biotechnology Co., Ltd.: using AAV9 plasmid as a vector, inserting a MYZAP interference fragment as shown in SEQ ID NO. 2 and an enhanced green fluorescent protein EGFP sequence, and using CTNT as a myocardial cell promoter to construct the recombinant vector for knocking down MYZAP gene expression. After the recombinant vector for knocking down MYZAP gene expression was successfully constructed, it was stored at -80°C for standby use. All virus-coated plasmids were verified by sequencing.

[0085] In order to verify the effect of knocking down MYZAP on the heart function of myocardial infarction mice, male C57BL / 6 mice (about 25 g) were injected with the recombinant vector for knocking down MYZAP gene expression AAV9-Sh-MYZAP through the tail vein, and the injection dose was 2×10 11 GC (genome containing particles, genome-containing particles) using normal saline (150 μl per mouse) as a solvent. The control group of mice was also injected with the same dose of adeno-associated virus carrying a negative control sequence (+NC) through the tail vein.

[0086] The mice were divided into a WT wild type group, a Sh-MYZAP knocking down group, and a NC negative control group, and after 4 weeks of injection, echocardiography was performed on the mice in each group to detect the heart function of the mice. The echocardiography detection method was the same as in Example 2.

[0087] The results are shown in Table 2. Figures 19-22 As shown in Table 2, after infection with the recombinant virus AAV9-Sh-MYZAP, the EF% and FS% values of the heart of the mice in the knocking down group were significantly reduced compared with the NC group, the LVIDd and LVIDs were significantly increased, and the LVPWd / LVPWs did not change significantly, which indicated that knocking down MYZAP can cause certain damage to the heart function of mice.

[0088] Example 6

[0089] In this embodiment, a recombinant vector for knocking down MYZAP gene expression, an adeno-associated virus vector AAV9-Sh-MYZAP, was constructed, and it was verified through in vivo experiments that knocking down MYZAP gene can significantly promote the occurrence of arrhythmia in mice.

[0090] The mice in each group were subjected to programmed electrical stimulation based on the mouse model of different groups in Example 5, and the incidence of ventricular arrhythmia after MI was detected. The specific method of programmed electrical stimulation was the same as that in Example 3.

[0091] The results are shown in Figures 23-25 Compared with the NC group, the incidence of programmed electrical stimulation-induced ventricular arrhythmia was significantly increased in the MYZAP knockdown mice, and the ventricular arrhythmia induction time was significantly prolonged. MYZAP silencing promoted the susceptibility and persistence of ventricular arrhythmia.

[0092] Example 7

[0093] In this embodiment, a recombinant vector for knocking down MYZAP gene expression, an adeno-associated virus vector AAV9-Sh-MYZAP, was constructed, and it was verified through in vivo experiments that knocking down MYZAP gene can significantly slow down the conduction of mouse heart and prolong APD.

[0094] In this embodiment, the changes of heart conduction velocity and APD of mice in each group were detected by Optical mapping based on the mouse model of different groups in Example 5. The specific method of Optical mapping detection was the same as that in Example 4.

[0095] The results are shown in Figures 26-32 Compared with the NC group, the heart conduction velocity of the MYZAP knockdown group of mice was significantly slowed down, while there was no significant difference between the NC group and the WT group. Compared with the NC group, the rate of action potential rise of the MYZAP knockdown mice was reduced, and the peak value was also reduced. Moreover, the ventricular APD 30 and APD 50 of the MYZAP knockdown group of mice were significantly prolonged. It was thus proved that the electrical conduction of the heart of the MYZAP knockdown mice was impaired.

Claims

1. The application of MYZAP overexpression recombinant vector in the preparation of drugs for the prevention and treatment of arrhythmia, characterized in that, The arrhythmia is a ventricular arrhythmia following myocardial infarction, and the nucleotide sequence of the MYZAP gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The MYZAP overexpression recombinant vector is an adeno-associated virus vector AAV9-MYZAP that overexpresses the MYZAP gene.

3. The application according to claim 2, characterized in that, The aforementioned antiarrhythmic drugs reduce the occurrence of arrhythmias after myocardial infarction by increasing the content of MYZAP protein in cardiac cardiomyocytes, and improve cardiac function and cardiac electrical conduction disorders after myocardial infarction.

Images