Application of MYZAP overexpression vector in the preparation of drugs for the prevention and treatment of MIRI

Abstract

This invention relates to the application of MYZAP overexpression vectors in the preparation of drugs for the prevention and treatment of myocardial ischemia-reperfusion injury (MIRI), belonging to the field of biomedical technology. To address the limitation of gene therapy applications in the prevention and treatment of myocardial ischemia-reperfusion injury, this invention provides the application of a recombinant vector overexpressing the myocardial band adhesion protein MYZAP in the preparation of drugs for the prevention and treatment of myocardial ischemia-reperfusion injury. The MYZAP gene overexpression recombinant vector is an adeno-associated virus vector AAV9-MYZAP that overexpresses MYZAP. Experiments have demonstrated that MYZAP overexpression can improve cardiac function after myocardial ischemia-reperfusion, reduce the incidence of arrhythmias, and improve the function of sodium, potassium, and calcium ion channels in cardiomyocytes. Based on this, this invention also provides a drug for the prevention and treatment of myocardial ischemia-reperfusion injury, offering a new strategy for gene therapy of myocardial ischemia-reperfusion injury with broad clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of MYZAP overexpression vector in the preparation of drugs for the prevention and treatment of MIRI. Background Art

[0002] Myocardial ischemia-reperfusion (I / R) aims to rescue ischemic cardiomyocytes, but it can also progressively worsen existing myocardial tissue damage, a phenomenon known as myocardial ischemia-reperfusion injury (MIRI). The severity of MIRI is influenced by various factors, including the duration of ischemia and the oxygen demand of the tissues and organs. During reperfusion, a large number of free radicals are generated, leading to oxidative stress; intracellular calcium overload causes arrhythmias and cardiomyocyte death; and the activation of the inflammatory response further damages the myocardium. This damage increases the infarct size, leads to heart failure, and increases the risk of arrhythmias.

[0003] Current prevention of myocardial ischemia-reperfusion injury (MIRI) involves timely adjustments to treatment plans, such as shortening ischemic time and optimizing reperfusion techniques. The use of calcium channel blockers, beta-blockers, and angiotensin-converting enzyme inhibitors reduces the occurrence of myocardial ischemic events. Pharmacological treatment of MIRI includes the use of anticoagulants, coronary vasodilators, antioxidants, anti-inflammatory drugs, and drugs that improve myocardial metabolism. Although strategies for preventing and treating MIRI are constantly evolving in clinical practice, there has been no satisfactory progress in significantly reducing the size of myocardial infarction or the incidence of arrhythmias.

[0004] Gene therapy can precisely target diseased genes and intervene at specific molecular targets. Its mechanism of action is well-defined, which helps reduce potential side effects. However, gene therapy is not yet widely used in the prevention and treatment of myocardial ischemia-reperfusion injury, mainly because its pathophysiological mechanism involves the complex regulation of multiple genes and signaling pathways, making it difficult to identify and validate effective therapeutic targets, thus limiting the precise selection of gene targets. Summary of the Invention

[0005] To address the limitation of gene therapy applications in the prevention and treatment of myocardial ischemia-reperfusion injury, this invention provides the application of the MYZAP overexpression vector in the preparation of drugs for the prevention and treatment of MIRI.

[0006] The technical solution of this invention:

[0007] This invention provides the application of the Myocardial zonula adherens protein (MYZAP) overexpression vector in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury.

[0008] Furthermore, the MYZAP overexpression vector is an adeno-associated virus vector AAV9-MYZAP that overexpresses the MYZAP gene.

[0009] Furthermore, the nucleotide sequence of the MYZAP gene is shown in SEQ ID NO.1.

[0010] Furthermore, the drug for preventing myocardial ischemia-reperfusion injury improves cardiac function after myocardial ischemia-reperfusion by increasing the content of MYZAP protein in cardiac cardiomyocytes, and improves the function of sodium ion channels, potassium ion channels and calcium ion channels in cardiomyocytes.

[0011] A drug for preventing myocardial ischemia-reperfusion injury, the drug containing a MYZAP gene overexpression vector, the nucleotide sequence of the MYZAP gene being shown in SEQ ID NO.1.

[0012] Furthermore, the MYZAP gene overexpression vector is an adeno-associated virus vector AAV9-MYZAP that overexpresses the MYZAP gene.

[0013] The beneficial effects of this invention are:

[0014] This invention targets the myocardial band adhesion protein MYZAP gene, constructing a recombinant vector for MYZAP gene overexpression mediated by adeno-associated virus (AAV) serotype 9—AAV9 and a mouse model of myocardial ischemia-reperfusion injury. In vivo and in vitro experiments confirmed that MYZAP overexpression can improve cardiac function after myocardial ischemia-reperfusion, reduce the incidence of arrhythmias, and improve the function of sodium, potassium, and calcium ion channels in cardiomyocytes. Based on this, this invention provides the application of the MYZAP gene as a drug target in the preparation of drugs to prevent and treat myocardial ischemia-reperfusion injury, offering a new strategy for gene therapy of myocardial ischemia-reperfusion injury with broad clinical application prospects. Attached Figure Description

[0015] Figure 1 The images show the electrocardiograms of mice in the Sham group, myocardial ischemia group, and reperfusion group in Example 1. A represents the Sham group, B represents the myocardial ischemia group, and C represents the reperfusion group.

[0016] Figure 2 This is a comparison of MYZAP mRNA levels in cardiomyocytes of mice in the Sham group and I / R group in Example 1.

[0017] Figure 3 The image shows the WB results of MYZAP expression in cardiomyocytes of mice in the Sham group and I / R group in Example 1. A is the Sham group and B is the I / R group.

[0018] Figure 4 This is a comparison of the MYZAP protein expression levels in cardiomyocytes of mice in the Sham group and the I / R group in Example 1;

[0019] Figure 5 Echocardiograms of mice in each group in Example 2: A is the Sham group, B is the I / R group, C is the +MYZAP group, and D is the +NC group.

[0020] Figure 6 This is a comparison chart of ejection fraction (EF) and left ventricular fractional shortening (FS) of mice in each group in Example 2. A represents EF and B represents FS.

[0021] Figure 7 The following are the electrocardiograms of mice in each group in Example 3: A is the Sham group, B is the I / R group, C is the +MYZAP group, and D is the +NC group.

[0022] Figure 8 This is a comparison chart of the incidence of ventricular arrhythmias in each group of mice in Example 3;

[0023] Figure 9 This is a comparison diagram of ventricular activity in each group of mice in Example 3;

[0024] Figure 10 Na+ in the mouse cardiomyocytes of each group in Example 4 v 1.5 WB results of protein expression: A is the Sham group, B is the I / R group, C is the +MYZAP group, and D is the +NC group;

[0025] Figure 11 Na+ in the mouse cardiomyocytes of each group in Example 4 v Figure 1.5 shows a comparison of protein expression levels and mRNA levels, where A represents protein expression level and B represents mRNA level.

[0026] Figure 12 I in each group of mouse cardiomyocytes in Example 4 Na Current density representation diagrams: A is the Sham group, B is the I / R group, C is the +MYZAP group, and D is the +NC group.

[0027] Figure 13 I in each group of mouse cardiomyocytes in Example 4 Na Current density peak value representation graph;

[0028] Figure 14 I in each group of mouse cardiomyocytes in Example 4Na Current density statistical comparison chart;

[0029] Figure 15 I in each group of mouse cardiomyocytes in Example 4 Na Statistical comparison chart of peak current density;

[0030] Figure 16 K in each group of mouse cardiomyocytes in Example 5 v 4.2 WB results of protein expression: A is the Sham group, B is the I / R group, C is the +MYZAP group, and D is the +NC group;

[0031] Figure 17 K in each group of mouse cardiomyocytes in Example 5 v Figure 4.2 shows a comparison of protein expression levels and mRNA levels, where A represents protein expression level and B represents mRNA level.

[0032] Figure 18 I in each group of mouse cardiomyocytes in Example 5 to Current density representation diagrams: A is the Sham group, B is the I / R group, C is the +MYZAP group, and D is the +NC group.

[0033] Figure 19 I in each group of mouse cardiomyocytes in Example 5 to Current density statistical comparison chart;

[0034] Figure 20 I in each group of mouse cardiomyocytes in Example 5 to Statistical comparison chart of peak current density;

[0035] Figure 21 For Ca in the mouse cardiomyocytes of each group in Example 6 v 1.2 WB results of protein expression: A is the normal cell group, B is the MYZAP knockdown group, and C is the NC negative control group;

[0036] Figure 22 For Ca in the mouse cardiomyocytes of each group in Example 6 v Figure 1.2 shows a comparison of protein expression levels and mRNA levels, where A represents protein expression level and B represents mRNA level.

[0037] Figure 23 I in each group of mouse cardiomyocytes in Example 6 Ca，L Current density representation graph: A represents normal cell group, B represents MYZAP knockdown group, and C represents NC negative control group;

[0038] Figure 24 I in each group of mouse cardiomyocytes in Example 6 Ca，L Current density statistical comparison chart;

[0039] Figure 25 I in each group of mouse cardiomyocytes in Example 6 Ca，L Current density peak statistics. Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0041] Example 1

[0042] This example demonstrates that the expression of the MYZAP gene is reduced in myocardial tissue after myocardial ischemia-reperfusion in mice.

[0043] This embodiment first constructs a mouse myocardial ischemia-reperfusion (I / R) model, and the specific construction method is as follows:

[0044] C57BL / 6 mice were anesthetized by intraperitoneal injection of afotin (0.2 g / kg). After anesthesia, the chest was shaved, and the mice were fixed in a supine position. A surface electrocardiogram (ECG) device was connected to the limbs to record the ECG. The mice were connected to a ventilator via their trachea, and their respiratory rate was synchronized with the ventilator's rate. Once synchronized, the skin on the left chest of the mice was cut open, and the muscle tissue was bluntly dissected. The thoracic cavity was opened between the second and third ribs to expose the heart. A 7-0 ligation suture was clamped with a needle holder, and the left anterior descending coronary artery was ligated 2 mm below the lower edge of the left atrial appendage. ECG changes were observed. ST segment elevation indicated correct ligation and successful ischemia. After 45 minutes of ischemia, the ligation suture was slowly released to restore coronary blood flow. The ST segment return indicated successful reperfusion. Reperfusion lasted for 24 hours, during which the mice's vital signs were continuously monitored. The Sham control group underwent only thoracotomy without ligation, and other treatments were the same as the model group.

[0045] Mice were subjected to 45 minutes of ischemia followed by 24 hours of reperfusion for subsequent experiments. qRT-PCR and Western blot experiments were performed on samples taken from the area below the cardiac ligation suture in mice to detect changes in relevant indicators.

[0046] (1) qRT-PCR experiment

[0047] Mouse left ventricular tissue was washed with pre-cooled PBS buffer, placed on filter paper, blotted dry with PBS, and then placed in a 1.5 ml EP tube. 300 µl of TRIzol was added to the EP tube, and the tissue was ground with an electric grinder until no obvious tissue blocks were found. Then, 700 µl of TRIzol was added.

[0048] Add 200 µl of chloroform to each sample and vortex vigorously for 15 seconds. Let stand for 10 minutes. Centrifuge at 4°C, 13500 rpm for 15 minutes. Slowly aspirate the supernatant into a new 1.5 ml EP tube, then add an equal volume of isopropanol to each tube, mix well, and let stand for 30 minutes. Centrifuge at 4°C, 13500 rpm for 10 minutes. Discard the supernatant, add 1000 µl of a mixture of anhydrous ethanol and DEPC water (3:1 ratio) to wash, gently agitate up and down; a feather-like precipitate will be observed in the tube. Centrifuge at 4°C, 10600 rpm for 5 minutes. Discard the supernatant, and add an appropriate amount of DEPC water to the EP tube to dissolve the RNA. After the RNA precipitate had completely dissolved, the RNA concentration was measured using a Nano Drop 8000 spectrophotometer. The RNA sample was then reverse transcribed into cDNA using a Toyobo reverse transcription kit for subsequent qRT-PCR amplification experiments.

[0049] (2) Western blot experiment

[0050] Western blot experiments were performed on cardiac cardiomyocytes from C57BL / 6 mice in the Sham and I / R groups. 150 μl of pre-prepared cell lysis buffer was added to the cells to be extracted (the amount of lysis buffer can be adjusted according to cell density), and the cells were incubated on ice for 5 minutes for lysis. Adherent cells were scraped off with a cell scraper, and the protein lysis buffer was collected into a 1.5 ml EP tube using a pipette. The cells were then centrifuged at 13500 rpm at 4°C for 15-20 minutes. After centrifugation, the supernatant was transferred to a new 1.5 ml EP tube; the supernatant was the extracted total protein. The prepared gel was first placed in the electrophoresis tank, and electrophoresis buffer was added to the appropriate mark. Protein samples were loaded in the order required for the experiment, followed by protein markers. The electrophoresis apparatus was connected, and the stacking gel voltage was adjusted to 70 V. Electrophoresis was run for approximately 30 minutes. Once the protein samples had passed through the stacking gel and formed a straight line, the voltage was adjusted to 110 V. Remove the gel using a gel release plate and place it in the following order: white sponge - filter paper - NC membrane - gel - filter paper - black sponge, gently removing any air bubbles. Keep the transfer tank at a low temperature (300 mA) and transfer for 120 minutes. Place the NC membrane in a pre-prepared blocking buffer containing 10% skim milk and block on a shaker at room temperature for 1.5 hours. Cut the target band from the blocked NC membrane according to the molecular weight of the target protein and place it in a pre-diluted primary antibody solution in a resealable bag for overnight incubation at 4°C. Remove the NC membrane and wash four times with PBST for 7 minutes each time, followed by incubation with a diluted secondary antibody solution at room temperature for 50 minutes, then develop and analyze.

[0051] The results are as follows Figures 2-4 As shown, qRT-PCR results revealed a significant decrease in MYZAP levels in the hearts of I / R mice (P < 0.05, n = 8). Western blot results were consistent with qRT-PCR results. The experimental results in this example demonstrate that MYZAP expression is reduced after myocardial ischemia-reperfusion injury.

[0052] Example 2

[0053] In this embodiment, a recombinant vector for overexpressing the MYZAP gene—the adeno-associated virus vector AAV9-MYZAP—was constructed, and in vivo experiments confirmed that overexpression of the MYZAP gene can significantly improve cardiac function in I / R mice.

[0054] Adeno-associated virus (AAV) is an effective and safe transduction vector. Different serotypes of AAV have different infectivity and gene delivery efficiency in tissues. In this embodiment, the AAV9 serotype with the highest infectivity in myocardial tissue was selected.

[0055] The construction of the MYZAP gene overexpression plasmid and the packaging of adeno-associated virus (AAV) were completed by Guangzhou Aizhe Biotechnology Co., Ltd. Using the AAV9 plasmid as a vector, the full-length MYZAP nucleotide sequence (as shown in SEQ ID NO.1) and the enhanced green fluorescent protein (EGFP) sequence were inserted, with CMV as a universal promoter for various cell types. After successful construction of the MYZAP gene overexpression recombinant vector, it was stored at -80℃ for later use. All virus-coated plasmids were sequenced for verification.

[0056] To confirm the effect of MYZAP overexpression on cardiac function in I / R mice, male C57BL / 6 mice (approximately 25 g) were injected via tail vein with the MYZAP gene overexpression recombinant vector AAV9-MYZAP at a dose of 2 × 10⁻⁶ per mouse. 11 GC (Genome containing particles) was administered in physiological saline (150 µl per mouse) as the solvent. Control mice were also injected via tail vein with the same dose of adeno-associated virus carrying the negative control sequence (+NC).

[0057] Six weeks after injection, the I / R model was established and divided into the Sham sham operation group, the I / R model group, the +MYZAP overexpression group (I / R+MYZAP), and the +NC negative control group (I / R+NC). Echocardiography was performed on mice in each group to detect cardiac function.

[0058] The methods for cardiac ultrasound examination are as follows:

[0059] Mice were weighed and anesthetized with an intraperitoneal injection of the anesthetic azedarach. They were then fixed in a supine position on a 37°C constant-temperature operating platform, their limbs secured with tape, and coupling gel applied to their chests after hair removal. M-Mode curves at the papillary muscle level of the left ventricular long axis and short axis were measured. Various cardiac parameters were determined using instrumental analysis software and analyzed using a VINNO 6 high-resolution imaging system. Ejection fraction (EF%) and fractional shortening (FS%) were calculated to assess cardiac function.

[0060] The results are as follows Figures 5-6 As shown, compared with the Sham group, the EF% and FS% of I / R group mice were significantly decreased, indicating weakened cardiac function. However, after AAV9-MYZAP treatment, EF% and FS% significantly increased. This suggests that MYZAP overexpression can effectively improve cardiac function in I / R mice.

[0061] Example 3

[0062] In this embodiment, a recombinant vector for overexpressing the MYZAP gene—the adeno-associated virus vector AAV9-MYZAP—was constructed, and in vivo experiments confirmed that overexpression of the MYZAP gene can significantly reduce the occurrence of arrhythmias in I / R mice.

[0063] Male C57BL / 6 mice (approximately 25 g) were injected via tail vein with the MYZAP gene overexpression recombinant vector AAV9-MYZAP at a dose of 2 × 10⁻⁶ per mouse. 11 GC (genome-containing particles) was administered in saline (150 µl per mouse) as the solvent. Control mice were injected intravenously via the tail vein with the same dose of adeno-associated virus carrying the negative control sequence (+NC).

[0064] Four weeks after injection, an I / R model was established and the mice were divided into a Sham sham-operated group, an I / R model group, a +MYZAP overexpression group (I / R+MYZAP), and a +NC negative control group (I / R+NC). The mice in each group were subjected to programmed electrical stimulation to induce arrhythmias, and the incidence of arrhythmias was detected.

[0065] The specific methods of programmed electrical stimulation are as follows:

[0066] After anesthetizing the mice, they were fixed supine under a stereomicroscope. The skin was incised along the right side of the neck, the glands were bluntly dissected to expose the jugular vein, and a small incision was made along the right branch of the vein near the head. An electrode was inserted anteriorly into the right ventricle of the mouse. A stimulation pattern of 10 consecutive electrical pulses (S1) with a coupling interval of 80 ms was used, followed by two additional stimuli (S2 and S3) at 80 and 78 ms respectively, with a coupling interval of 2 ms, decreasing in step size of -2. The occurrence of arrhythmias was recorded at three voltages: 3.5V, 5V, and 8V. The waveform was adjusted, and the stimulation program was started, thus inducing ventricular arrhythmias. The stimulation was repeated twice to check the reproducibility of the experiment.

[0067] The results are as follows Figure 7-Figure 9 As shown, compared with the Sham group, the incidence of arrhythmia in the I / R group mice was significantly increased, while AAV9-MYZAP treatment significantly reversed the increase in the incidence of arrhythmia; compared with the Sham group, the induction duration of arrhythmia in the I / R group mice was significantly prolonged, while AAV9-MYZAP treatment significantly reversed the prolonged induction duration of arrhythmia. This indicates that MYZAP overexpression can reduce the incidence and induction duration of arrhythmia in I / R mice, effectively improving arrhythmia after I / R.

[0068] Example 4

[0069] In this embodiment, a recombinant vector for overexpressing the MYZAP gene—the adeno-associated virus vector AAV9-MYZAP—was constructed, and the effect of overexpressing the MYZAP gene on sodium ion channels in I / R mice was confirmed through in vivo experiments.

[0070] Male C57BL / 6 mice (approximately 25 g) were injected via tail vein with the MYZAP gene overexpression recombinant vector AAV9-MYZAP at a dose of 2 × 10⁻⁶ per mouse. 11 GC (genome-containing particles) were administered in saline (150 µl per mouse) as the solvent. Control mice were also injected via tail vein with the same dose of adeno-associated virus carrying the negative control sequence (+NC).

[0071] Four weeks after injection, an I / R model was established, and participants were divided into a Sham sham-operated group, an I / R model group, a +MYZAP overexpression group (I / R+MYZAP), and a +NC negative control group (I / R+NC). Na+ was detected using Western blotting and qRT-PCR. v 1.5 Changes in protein and mRNA expression.

[0072] Patch clamp technique for detecting sodium channel current I Na The specific method is as follows:

[0073] Cardiac cardiomyocytes were placed in a bath, and a prepared extracellular solution was added. A glass microelectrode was drawn, and then the electrode fluid used to record the sodium current was infused to two-thirds of the electrode. The electrode was then connected to the probe of a patch-clamp instrument. The cell state was observed under an inverted microscope. The electrode was gently moved by micromanipulation to make slight contact with the cell membrane surface, and an appropriate negative pressure was applied to seal the cells. A high-resistance seal was formed when the display resistance reached 1 GΩ. At this point, the membrane was ruptured by applying negative pressure through appropriate aspiration. After rupture, an appropriate stimulation program was selected to record the ion current, and the data was saved for analysis.

[0074] The results are as follows Figures 10-15 As shown, Western blot results indicate that I / R mice have Na v 1.5 Protein expression levels were significantly reduced after AAV9-MYZAP treatment with Na v 1.5 Protein expression levels were significantly increased; qRT-PCR results showed that Na in I / R mice was significantly elevated. v 1.5 mRNA expression levels were significantly reduced after AAV9-MYZAP treatment with Na v 1.5 mRNA expression levels were significantly increased. Whole-cell patch-clamp results showed that I / R mice I Na Significantly reduced, I after AAV9-MYZAP treatment Na Increased; peak I in I / R mice NaThe peak I value was significantly reduced after AAV9-MYZAP treatment. Na Increase.

[0075] This embodiment demonstrates that MYZAP overexpression can increase Na+ in cardiomyocytes of I / R mice. v 1.5 Protein and mRNA expression levels can increase sodium ion current I in I / R mouse cardiomyocytes. Na And increase peak I Na This indicates that MYZAP has a regulatory effect on sodium ion channels in cardiomyocytes.

[0076] Example 5

[0077] In this embodiment, a recombinant vector for overexpressing the MYZAP gene—the adeno-associated virus vector AAV9-MYZAP—was constructed, and the effect of overexpressing the MYZAP gene on potassium ion channels in I / R mice was confirmed through in vivo experiments.

[0078] Male C57BL / 6 mice (approximately 25 g) were injected via tail vein with the MYZAP gene overexpression recombinant vector AAV9-MYZAP at a dose of 2 × 10⁻⁶ per mouse. 11 GC (genome-containing particles) were administered in saline (150 µl per mouse) as the solvent. Control mice were also injected via tail vein with the same dose of adeno-associated virus carrying the negative control sequence (+NC).

[0079] Four weeks after injection, an I / R model was established, and participants were divided into a Sham sham-operated group, an I / R model group, a +MYZAP overexpression group (I / R+MYZAP), and a +NC negative control group (I / R+NC). K+ was detected using Western blotting and qRT-PCR techniques. v 4.2 Changes in protein and mRNA expression. Patch-clamp technique was used to detect transient outward potassium current I. to .

[0080] The results are as follows Figures 16-20 As shown, Western blot results indicate that I / R mice K v 4.2 Protein expression levels were significantly reduced after AAV9-MYZAP treatment with K v 4.2 Protein expression levels were significantly increased; qRT-PCR results showed that K in I / R mice was significantly elevated. v 4.2 mRNA expression levels were significantly reduced after AAV9-MYZAP treatment with K v 4.2 mRNA expression levels were significantly increased. Whole-cell patch-clamp results showed that I / R mice with I... to Significantly reduced, I after AAV9-MYZAP treatment to Increased; I / R mice I toThe peak value was significantly reduced after AAV9-MYZAP treatment. to The peak value increases.

[0081] This embodiment demonstrates that MYZAP overexpression can increase the K+ level in cardiomyocytes of I / R mice. v 4.2 Protein and mRNA expression levels can increase the potassium ion current I in I / R mouse cardiomyocytes. to And increase I to Peak value. This indicates that MYZAP has a regulatory effect on potassium ion channels in cardiomyocytes.

[0082] Example 6

[0083] In this embodiment, a MYZAP gene knockdown sequence (si-MYZAP) was constructed, and the effect of knocking down the MYZAP gene on calcium ion channels in normal mice was confirmed by in vitro experiments.

[0084] Using newborn Kunming mice (1-3 days old), disinfect their entire bodies by immersing them in 75% alcohol. After decapitation, cut open the chest and remove the heart with curved forceps. Place the heart in a glass dish containing an appropriate amount of PBS buffer and 0.5 ml of a penicillin-streptomycin-gentamicin mixture. After the heart is completely removed, use straight forceps to transfer it to another glass dish containing the same PBS buffer and 0.5 ml of penicillin-streptomycin-gentamicin mixture. Then transfer it to a 50 ml centrifuge tube, discard the original liquid, add 2-3 ml of D-Hanks, gently pipette to wash away blood clots, and repeat 2-3 times. After draining the blood, add 3 ml of D-Hanks and 2 ml of trypsin (enough for 30 mice) to the 50 ml centrifuge tube, gently pipette to mix, seal, and digest overnight on a shaker at 4°C for 8-12 hours.

[0085] After digestion, discard the digestion solution in the centrifuge tube and add DMEM culture medium containing 10% fetal bovine serum to terminate digestion. Then, using a 50 ml sterile syringe and a 0.22 μm microporous membrane, mix 16.8 mg of type II collagenase and 21 ml of DMEM (enough for 30 suckling mice), filter, and incubate at 37°C for 15 minutes. After discarding the culture medium, add 6 ml of the type II collagenase and DMEM mixture, and incubate at 37°C on a shaker for 12 minutes. Transfer the supernatant to another 50 ml centrifuge tube and repeat the digestion 4-5 times until the heart size is significantly reduced. Centrifuge at 1500 rpm for 5 minutes. After centrifugation, slowly discard the supernatant, add DMEM culture medium containing 10% fetal bovine serum to the pellet, gently pipette to disperse the cells evenly, and then transfer to a culture flask. Incubate for 1 hour to separate cardiomyocytes from fibroblasts. One hour later, the fibroblasts had almost adhered to the bottle wall. The culture medium containing cardiomyocytes was collected into a new culture bottle, spread evenly, and the cardiomyocytes were spread in a cell culture plate and cultured in an incubator for 48 hours.

[0086] Observe the state of cardiomyocytes after 48 hours of culture under a microscope. If the cardiomyocytes have basically completely adhered to the culture plate and are growing well, cell transfection can be performed. Transfect the cells with si-MYZAP (MYZAP knockdown sequence) and NC sequences as shown in SEQ ID NO.2. Taking one well of a six-well plate as an example, the specific operation is as follows: After turning off the light source in the clean bench, prepare solution A: Mix 100 μl of opti transfection reagent with 8 μl of X-treme in a light-proof tube and let stand for 5 minutes. Prepare solution B: Mix 100 μl of opti transfection reagent with 100 μg of si-MYZAP and NC in light-proof tubes and let stand for 5 minutes. Discard the original liquid in the culture plate, add DMEM, mix solution A and solution B evenly, let stand for 20 minutes, and finally add the prepared liquid to each well of the culture plate. The amount of transfection reagent needs to be determined based on the number of cells. The transfection time is 12 hours. After transfection, the liquid should be replaced with DMEM culture medium containing 10% fetal bovine serum.

[0087] Primary cardiomyocytes from neonatal mice were isolated and divided into Ctl (normal cells) group, si-MYZAP group, and NC negative control group. Ca2+ was detected by Western blotting and qRT-PCR, respectively. v 1.2 Changes in protein and mRNA expression. Patch-clamp technique was used to detect L-type calcium channel current I. Ca，L .

[0088] The results are as follows Figures 21-25As shown, Western blot results indicate that, compared to the NC group, knocking down MYZAP resulted in a decrease in Ca2+ levels. v 1.2 Increased protein expression; qRT-PCR results showed that, compared with the NC group, knockdown of MYZAP significantly reduced Ca2+ expression. v 1.2 Increased mRNA expression levels. Whole-cell patch-clamp results showed that, compared with the NC group, knockdown of MYZAP significantly increased mRNA expression. Ca，L Increased; compared with the NC group, knocking down MYZAP resulted in increased I Ca，L The peak value increased significantly.

[0089] This embodiment demonstrates that MYZAP knockdown can increase calcium levels in neonatal mouse cardiomyocytes. v 1.2 Protein and mRNA expression levels can increase the calcium ion current I in neonatal mouse cardiomyocytes. Ca，L And increase I Ca，L Peak value. This indicates that MYZAP has a regulatory effect on calcium ion channels in cardiomyocytes.

Claims

1. The application of the MYZAP overexpression recombinant vector in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury, characterized in that, 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 drug for preventing myocardial ischemia-reperfusion injury improves cardiac function after myocardial ischemia-reperfusion by increasing the content of MYZAP protein in cardiac cardiomyocytes, and improves the function of sodium ion channels, potassium ion channels and calcium ion channels in cardiomyocytes.

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