Application of MYZAP overexpression vector in preparation of medicine for preventing and treating MIRI

Abstract

The invention relates to application of an MYZAP overexpression vector in preparation of a medicine for preventing and treating MIRI, and belongs to the technical field of biological medicine. In order to solve the problem that application of gene drugs in prevention and treatment of myocardial ischemia reperfusion injury is limited, the invention provides application of an MYZAP gene overexpression recombinant vector in preparation of drugs for prevention and treatment of myocardial ischemia reperfusion injury, and the MYZAP gene overexpression recombinant vector is an adeno-associated virus vector AAV9-MYZAP for overexpressing MYZAP. Experiments prove that MYZAP overexpression can improve the cardiac function after myocardial ischemia reperfusion, reduce the occurrence rate of arrhythmia and improve the functions of sodium ion channels, potassium ion channels and calcium ion channels of myocardial cells. On the basis, the invention further provides a medicine for preventing and treating myocardial ischemia reperfusion injury, a new strategy is provided for gene therapy of myocardial ischemia reperfusion injury, and the medicine has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of a MYZAP overexpression vector in the preparation of a drug for preventing and treating MIRI. Background Art

[0002] Myocardial ischemia-reperfusion (I / R) aims to rescue ischemic myocardial cells, but it can also cause progressive damage to existing myocardial tissue, a phenomenon known as myocardial ischemia-reperfusion injury (MIRI). The severity of MIRI is influenced by multiple factors, including the duration of ischemia and the oxygen demand of tissues and organs. Reperfusion produces a large number of free radicals, leading to oxidative stress. Intracellular calcium overload can cause arrhythmias and cardiomyocyte death, and activation of the inflammatory response can further damage the myocardium. This damage can increase myocardial infarction size, lead to heart failure, and increase the risk of arrhythmias.

[0003] Current prevention of MIRI can be achieved by timely adjusting treatment plans by shortening ischemic time and optimizing reperfusion methods. Calcium channel blockers, beta-blockers, and angiotensin-converting enzyme inhibitors are used to reduce the occurrence of myocardial ischemic events. Drug treatment for MIRI includes the use of anticoagulants, coronary artery dilators, antioxidants, anti-inflammatory drugs, and drugs that improve myocardial metabolism. Although strategies for preventing and treating MIRI continue to evolve in clinical practice, there has been no satisfactory progress that can significantly reduce the size of myocardial infarction and the occurrence of arrhythmias.

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

[0005] In order to solve the problem of limited application of gene drugs in the prevention and treatment of myocardial ischemia-reperfusion injury, the present invention provides the use of a MYZAP overexpression vector in the preparation of drugs for the prevention and treatment of MIRI.

[0006] The technical solution of the present invention:

[0007] The present invention provides use of a myocardial zonula adherens protein MYZAP overexpression vector in the preparation of a drug 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 and treating myocardial ischemia-reperfusion injury improves cardiac function after myocardial ischemia-reperfusion by increasing the content of MYZAP protein in cardiac myocardial cells, and improves the functions of sodium ion channels, potassium ion channels and calcium ion channels in cardiac myocardial cells.

[0011] A medicine for preventing and treating myocardial ischemia-reperfusion injury, comprising a MYZAP gene overexpression vector, wherein the nucleotide sequence of the MYZAP gene is 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] Beneficial effects of the present invention:

[0014] This invention uses the zonular adhesion protein MYZAP gene as a drug target, constructing an adeno-associated virus (AAV) serotype 9 (AAV9)-mediated MYZAP gene overexpression recombinant vector 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 channels in myocardial cells. Based on this, the invention provides the use of the MYZAP gene as a drug target in the preparation of drugs for the prevention and treatment of myocardial ischemia-reperfusion injury, providing a new strategy for gene therapy of myocardial ischemia-reperfusion injury and promising broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the electrocardiograms of the sham group, myocardial ischemia mice, and reperfusion mice in Example 1, A for the sham group, B for the myocardial ischemia group, and C for 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 Figure 1 shows the Western blot results of MYZAP expression in cardiomyocytes of mice in the sham group and the 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 MYZAP protein expression levels in cardiomyocytes of mice in the Sham group and I / R group in Example 1;

[0019] Figure 5 These are the 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 The figure is a comparison of the ejection fraction EF and the left ventricular fractional shortening FS of each group of mice in Example 2, A is EF, and B is FS;

[0021] Figure 7 These 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 of the chamber properties of each group of mice in Example 3;

[0024] Figure 10 is the Na in the myocardial cells of each group of mice in Example 4 v 1.5 Protein expression Western blotting results, 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 is the Na in the myocardial cells of each group of mice in Example 4 v Comparison of protein expression level and mRNA level of 1.5, A is protein expression level, B is mRNA level;

[0026] Figure 12 For each group of mouse myocardial cells in Example 4 Na Representative graphs of current density, 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 For each group of mouse myocardial cells in Example 4 Na Representative graph of current density peak;

[0028] Figure 14 For each group of mouse myocardial cells in Example 4Na Current density statistical comparison chart;

[0029] Figure 15 For each group of mouse myocardial cells in Example 4 Na Current density peak statistical comparison chart;

[0030] Figure 16 is the K in the myocardial cells of each group of mice in Example 5 v 4.2 Protein expression WB results, 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 is the K in the myocardial cells of each group of mice in Example 5 v Comparison of protein expression levels and mRNA levels of 4.2, A is the protein expression level, B is the mRNA level;

[0032] Figure 18 For each group of mouse myocardial cells in Example 5 to Representative graphs of current density, 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 For each group of mouse myocardial cells in Example 5 to Current density statistical comparison chart;

[0034] Figure 20 For each group of mouse myocardial cells in Example 5 to Current density peak statistical comparison chart;

[0035] Figure 21 is the Ca in the myocardial cells of each group of mice in Example 6 v 1.2 Protein expression WB results, A is the normal cell group, B is the MYZAP knockdown group, and C is the NC negative control group;

[0036] Figure 22 is the Ca in the myocardial cells of each group of mice in Example 6 v Comparison of protein expression level and mRNA level of 1.2, A is protein expression level, B is mRNA level;

[0037] Figure 23 For each group of mouse myocardial cells in Example 6 Ca，L Representative graphs of current density, A is the normal cell group, B is the MYZAP knockdown group, and C is the NC negative control group;

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

[0039] Figure 25 For each group of mouse myocardial cells in Example 6 Ca，L Current density peak statistics. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples 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] In this example, a myocardial ischemia-reperfusion (I / R) model was first constructed in mice. The specific construction method is as follows:

[0044] C57BL / 6 mice were anesthetized with an intraperitoneal injection of avertin (0.2 g / kg). After anesthesia, chest hair was removed and the mice were fixed in the supine position. A surface electrocardiogram (ECG) was connected to each of their limbs to record the ECG. The mouse's trachea was connected to a respiratory apparatus, and its respiratory rate was synchronized with the ventilator's. Once synchronized, the skin on the left chest was incised, and the muscle tissue was bluntly dissected. The chest cavity was opened between the second and third ribs to expose the heart. A 7-0 ligature was applied with a needle holder to the left anterior descending coronary artery 2 mm below the inferior margin of the left atrial appendage. Electrocardiographic changes were observed. An elevation of the ST segment indicated correct ligature placement and successful ischemia. After 45 minutes of ischemia, the ligature was slowly loosened to restore coronary blood flow. A decrease in the ST segment indicated successful reperfusion. Reperfusion lasted for 24 hours, during which time the mice's vital signs were continuously monitored. The sham control group underwent thoracotomy without ligation. All other treatments were the same as those in the model group.

[0045] Mice were subjected to 45 minutes of ischemia and 24 hours of reperfusion, and samples were collected for subsequent experiments. The area below the ligature of the heart was harvested for qRT-PCR and Western blot analysis to detect changes in related indicators.

[0046] (1) qRT-PCR experiment

[0047] Wash the mouse left ventricular tissue with pre-cooled PBS buffer, place the tissue on filter paper, absorb the PBS, and place the washed tissue in a 1.5 ml EP tube. Add 300 µl TRIzol to the EP tube and grind with an electric grinder until no obvious tissue fragments are left. Then add another 700 µl TRIzol.

[0048] Add 200 µl of chloroform to each sample and shake vigorously for 15 seconds. Let stand for 10 minutes. Centrifuge at 4°C, 13,500 rpm, and 15 minutes. Gently aspirate the supernatant into a new 1.5 ml EP tube. Add an equal volume of isopropanol to each tube, mix, and let stand for 30 minutes. Centrifuge at 4°C, 13,500 rpm, and 10 minutes. Discard the supernatant and wash with 1000 µl of a mixture of anhydrous ethanol and DEPC water (3:1 ratio). Gently shake the tube until a feathery precipitate is observed. Centrifuge at 4°C, 10,600 rpm, and 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 was completely dissolved, the RNA concentration was measured using a Nano Drop 8000 spectrophotometer. The RNA sample was reverse transcribed into cDNA using a Toyobo reverse transcription kit for subsequent qRT-PCR amplification.

[0049] (2) Western blot experiment

[0050] Western blot analysis was performed on cardiac myocytes from C57BL / 6 mice in the sham and I / R groups. 150 μl of pre-prepared cell lysis buffer (the amount of lysis buffer added can be adjusted based on cell density) was added to the cells to be extracted. Lyse the cells on ice for 5 minutes. Adherent cells were scraped off with a cell scraper, and the protein lysate was collected using a pipette into a 1.5 ml EP tube. The cells were then centrifuged at 13,500 rpm at 4°C for 15-20 minutes. After centrifugation, the supernatant was aspirated into a new 1.5 ml EP tube. This constituted the total protein extracted. First, place the prepared gel in the electrophoresis tank and fill it to the appropriate mark with electrophoresis buffer. Load the protein samples in the order specified for the experiment, then add the protein marker. Connect the electrophoresis apparatus and adjust the stacking gel voltage to 70 V. Run the electrophoresis for approximately 30 minutes. Once the protein samples have passed through the stacking gel and formed a straight line, adjust the voltage to 110 V. Remove the gel using a gel remover and arrange the gel in the order of white sponge, filter paper, NC membrane, gel, filter paper, and black sponge, gently removing any air bubbles. Place the transfer chamber on ice to keep the temperature low and transfer the membrane at a constant current of 300 mA 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. Incubate overnight at 4°C in a ziplock bag. Remove the NC membrane and wash it four times with PBST for 7 minutes each. Then, incubate it with a diluted secondary antibody solution at room temperature for 50 minutes before developing and analyzing.

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

[0052] Example 2

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

[0054] Adeno-associated virus is an effective and safe transduction vector. Its different serotypes vary in tissue infection ability and gene delivery efficiency. In this example, the AAV9 serotype with the strongest infection efficiency in myocardial tissue was selected.

[0055] The MYZAP gene overexpression plasmid and adeno-associated virus (AAV) packaging were completed by Guangzhou Aizhe Biotechnology Co., Ltd. Using an AAV9 plasmid as the vector, the full-length MYZAP sequence (shown in SEQ ID NO. 1) and the enhanced green fluorescent protein (EGFP) sequence were inserted, using CMV as a universal promoter for various cell types. After successful construction, the MYZAP gene overexpression recombinant vector was stored at -80°C until use. All virally packaged plasmids were verified by sequencing.

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

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

[0058] Cardiac ultrasound testing methods are as follows:

[0059] Mice were weighed and anesthetized with an intraperitoneal injection of the anesthetic avertin. The mice were placed in a supine position on a 37°C thermostatted operating platform. The limbs were secured with tape. After hair removal, coupling agent was applied to the chest. Left ventricular long-axis and short-axis M-Mode curves were measured at the papillary muscle level. Cardiac parameters were determined using instrument analysis software and analyzed using a VINNO 6 high-resolution imaging system. Ejection fraction (EF%) and left ventricular fractional shortening (FS%) were calculated to assess cardiac function.

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

[0061] Example 3

[0062] In this example, a MYZAP gene overexpression recombinant vector, 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 (about 25 g) were injected with the MYZAP gene overexpression recombinant vector AAV9-MYZAP via the tail vein at a dose of 2 × 10 11 GC (genome containing particles) were injected with saline (150 µl per mouse). A control group of mice was also injected via the tail vein with the same dose of adeno-associated virus carrying a negative control sequence (+NC).

[0064] Four weeks after injection, the I / R model was established and the mice were divided into sham operation group, I / R model group, +MYZAP overexpression group (I / R+MYZAP) and +NC negative control group (I / R+NC). Programmed electrical stimulation was performed on the mice in each group to induce arrhythmia, and the incidence of arrhythmia was detected.

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

[0066] After anesthetizing the mouse, it was fixed in a supine position under a stereomicroscope. The skin was cut open along the right side of the neck, and the gland was bluntly dissected to expose the jugular vein. A small incision was made along the right branch of the blood vessel near the head. The electrode was inserted forward from this point and delivered into the right ventricle of the mouse. The stimulation mode used was 10 continuous electrical pulses (S1) with a coupling interval of 80 ms. Then two additional stimulations (S2 and S3) were applied for 80 and 78 ms, respectively, with a coupling interval of 2 ms, that is, decreasing in steps of -2. The occurrence of arrhythmias at three voltages, 3.5 V, 5 V, and 8 V, were recorded. The waveform was adjusted and the stimulation program was started. Ventricular arrhythmias were thus induced. The stimulation was repeated twice to check the reproducibility of the experiment.

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

[0068] Example 4

[0069] In this example, a MYZAP gene overexpression recombinant vector, adeno-associated virus vector AAV9-MYZAP, was constructed, and in vivo experiments were performed to confirm the effect of MYZAP gene overexpression on sodium ion channels in I / R mice.

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

[0071] Four weeks after injection, an I / R model was established and the cells were divided into sham operation group, I / R model group, +MYZAP overexpression group (I / R+MYZAP) and +NC negative control group (I / R+NC). Western Blot and qRT-PCR were used to detect Na v 1.5 Changes in protein and mRNA expression.

[0072] Patch clamp technique to detect sodium channel current I Na , the specific method is as follows:

[0073] Place the cardiomyocytes in a bath and add the prepared extracellular solution. Draw a glass microelectrode and perfuse the solution containing the sodium current to be recorded up to two-thirds of the way up the electrode. Then connect the electrode to the probe of the patch clamp instrument. Observe the cell under an inverted microscope. Use micromanipulation to gently move the electrode to the cell membrane surface. Apply a moderate negative pressure to seal the cell. A high-resistance seal is achieved when the displayed resistance reaches 1 GΩ. Then, apply negative pressure by suction to rupture the membrane. After rupture, select the appropriate stimulation program to record the ion current and save the data for analysis.

[0074] The results are as follows Figure 10-15 As shown, Western Blot results showed that Na v 1.5 protein expression was significantly reduced, and Na v 1.5 protein expression was significantly increased; qRT-PCR results showed that Na v 1.5mRNA expression was significantly decreased after AAV9-MYZAP treatment. v The expression of 1.5 mRNA was significantly increased. The whole-cell patch clamp results showed that I / R mice Na Significantly decreased after AAV9-MYZAP treatment Na Increased; Peak I in I / R mice NaSignificantly reduced, peak I after AAV9-MYZAP treatment Na Increase.

[0075] This example demonstrates that MYZAP overexpression can increase Na v 1.5 Protein expression and mRNA expression can increase the sodium ion current I in myocardial cells of I / R mice Na , and increase the peak value I Na This indicates that MYZAP has a regulatory effect on sodium ion channels in cardiomyocytes.

[0076] Example 5

[0077] In this example, a MYZAP gene overexpression recombinant vector, adeno-associated virus vector AAV9-MYZAP, was constructed, and in vivo experiments were performed to confirm the effect of MYZAP gene overexpression on potassium channels in I / R mice.

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

[0079] Four weeks after injection, an I / R model was established and the cells were divided into sham operation group, I / R model group, +MYZAP overexpression group (I / R+MYZAP) and +NC negative control group (I / R+NC). Western Blot and qRT-PCR were used to detect K 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 showed that I / R mice K v 4.2 Protein expression was significantly reduced after AAV9-MYZAP treatment. v 4.2 Protein expression was significantly increased; qRT-PCR results showed that K v 4.2 mRNA expression was significantly reduced after AAV9-MYZAP treatment. v 4.2 mRNA expression was significantly increased. Whole-cell patch clamp results showed that I / R mice I to Significantly decreased after AAV9-MYZAP treatment to Increased; I / R mice I toThe peak value was significantly reduced after AAV9-MYZAP treatment. to Peak value increased.

[0081] This example demonstrates that MYZAP overexpression can increase K v 4.2 Protein expression and mRNA expression can increase the potassium ion current I in myocardial cells of I / R mice to , and increase I to This indicates that MYZAP has a regulatory effect on potassium channels in cardiomyocytes.

[0082] Example 6

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

[0084] Newborn Kunming rats (1-3 days old) were disinfected by soaking their bodies in 75% alcohol. After decapitation, the thorax was cut open and the heart removed with curved forceps. The heart was placed in a glass dish containing an appropriate amount of PBS buffer and 0.5 ml of a penicillin-streptomycin-gentamicin mixture. Once the heart was completely removed, it was transferred using straight forceps to another glass dish containing an appropriate amount of PBS buffer and 0.5 ml of a penicillin-streptomycin-gentamicin mixture. The heart was then transferred to a 50 ml centrifuge tube. The remaining liquid was discarded, and 2-3 ml of D-Hanks solution was added to the tube, gently pipetting to remove blood clots. This was repeated 2-3 times. After exsanguination, 3 ml of D-Hanks solution and 2 ml of trypsin (for 30 rats) were added to the 50 ml centrifuge tube. Gently pipetting was performed to mix thoroughly, the tube was sealed, and digestion was carried out in a shaker at 4°C overnight. The digestion time was 8-12 hours.

[0085] At the end of digestion, discard the digestion solution in the centrifuge tube and add DMEM supplemented with 10% fetal bovine serum to terminate the digestion. Then, using a 50 ml sterile syringe and a 0.22 μm microporous filter, mix 16.8 mg of type II collagenase and 21 ml of DMEM (enough for 30 pups) and filter. Place the tube in a 37°C incubator for 15 minutes. After discarding the incubation solution, add 6 ml of the type II collagenase and DMEM mixture and incubate in a 37°C shaker for 12 minutes. Aspirate the supernatant and transfer it to another 50 ml centrifuge tube. Continue digesting the tube 4-5 times until the heart is noticeably smaller. Centrifuge at 1500 rpm for 5 minutes. After centrifugation, slowly discard the supernatant and add DMEM supplemented with 10% fetal bovine serum to the pellet. Gently pipette to evenly disperse the cells. Plate the pellet into a culture flask and incubate for 1 hour to separate the cardiomyocytes from the fibroblasts. After 1 hour, the fibroblasts were almost attached to the flask wall. The culture medium containing the cardiomyocytes was collected in a new culture flask, blown evenly, and the cardiomyocytes were plated in a cell culture plate and cultured in an incubator for 48 hours.

[0086] Observe the cardiomyocytes under a microscope after 48 hours of culture. If they have essentially completely adhered and are growing well, proceed with cell transfection. Transfect the cells with the si-MYZAP (MYZAP knockdown sequence) and NC sequences shown in SEQ ID NO. 2. Using one well of a six-well plate as an example, the following steps are 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, respectively, in a light-proof tube and let stand for 5 minutes. Discard the remaining solution in the culture plate, add DMEM, mix Solution A and Solution B thoroughly, let stand for 20 minutes, and finally, add the prepared solution to each well of the culture plate. The amount of transfection reagent needs to be determined according to the number of cells. The transfection time is 12 hours. After the transfection is completed, the liquid is replaced with DMEM culture medium containing 10% fetal bovine serum.

[0087] Primary cardiomyocytes from neonatal mice were isolated and divided into Ctl (normal cell) group, si-MYZAP group and NC negative control group. Western Blot and qRT-PCR were used to detect Ca 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 Figure 21-Figure 25As shown in the Western Blot results, compared with the NC group, the Ca v 1.2 Increased protein expression; qRT-PCR results showed that compared with the NC group, the Ca v 1.2 mRNA expression increased. Whole-cell patch clamp results showed that compared with the NC group, after knocking down MYZAP, I Ca，L Compared with the NC group, after knocking down MYZAP, I Ca，L The peak value increased significantly.

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

Claims

1. Application of MYZAP overexpression vector in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury.

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

3. The use according to claim 2, characterized in that The nucleotide sequence of the MYZAP gene is shown in SEQ ID NO.

1.

4. The use according to claim 3, characterized in that The drug for preventing and treating myocardial ischemia-reperfusion injury improves cardiac function after myocardial ischemia-reperfusion by increasing the content of MYZAP protein in cardiac myocardial cells, and improves the functions of sodium ion channels, potassium ion channels and calcium ion channels in cardiac myocardial cells.

5. A drug for preventing and treating myocardial ischemia-reperfusion injury, characterized in that: The drug contains a MYZAP gene overexpression vector, and the nucleotide sequence of the MYZAP gene is shown in SEQ ID NO.

1.

6. The drug for preventing and treating myocardial ischemia-reperfusion injury according to claim 5, characterized in that: The MYZAP gene overexpression vector is an adeno-associated virus vector AAV9-MYZAP that overexpresses the MYZAP gene.

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