Use of tdp-43 overexpression vector in preparation of drugs for preventing and treating arrhythmia after myocardial ischemia-reperfusion

Abstract

The application relates to application of a TDP-43 overexpression vector in preparation of a medicine for preventing and treating arrhythmia after myocardial ischemia reperfusion, and belongs to the technical field of biological medicine. In order to solve the problem of lack of effective target points for preventing and treating arrhythmia after myocardial ischemia reperfusion, the application provides application of a TDP-43 overexpression vector in preparation of a medicine for preventing and treating arrhythmia after myocardial ischemia reperfusion, wherein the TDP-43 overexpression vector is a recombinant vector containing a TDP-43 gene coding sequence. In-vivo experiments carried out with the aid of a mouse myocardial ischemia reperfusion model prove that overexpression of TDP-43 can obviously reduce the incidence of ventricular arrhythmia after myocardial ischemia reperfusion, shorten the duration of arrhythmia attack, improve the cardiac electric conduction speed, and improve the overall function of the heart. Research proves that a gene therapy strategy taking TDP-43 as a target point provides a new target point and gene therapy idea for prevention and treatment of arrhythmia after myocardial ischemia reperfusion.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of TDP-43 overexpression vector in the preparation of drugs for the prevention and treatment of arrhythmias after myocardial ischemia-reperfusion. Background Technology

[0002] Myocardial ischemia / reperfusion (I / R) refers to the process by which myocardial ischemia and hypoxia caused by coronary artery occlusion are restored through thrombolysis, interventional procedures, or surgery, allowing the ischemic myocardium to regain blood perfusion. I / R injury is a common complication after thrombolysis or interventional procedures following acute myocardial infarction. It can induce myocardial electrical remodeling, downregulation of gap junction protein Cx43, myocardial ion channel disorder, and decreased myocardial conduction velocity, leading to a high incidence of ventricular arrhythmias, decreased cardiac function, and in severe cases, heart failure.

[0003] Currently, the main drugs used clinically for arrhythmias after myocardial ischemia-reperfusion include calcium channel blockers, beta-blockers, sodium channel blockers, and drugs that prolong the action potential duration. Although these drugs have certain efficacy in treating arrhythmias after myocardial ischemia-reperfusion, they also have some side effects. Some antiarrhythmic drugs may inhibit the normal contractile function of the heart, leading to a decrease in cardiac output, which may cause heart failure in severe cases; they have a vasodilatory effect, which may lead to a drop in blood pressure, causing symptoms such as dizziness and fatigue; some drugs may cause irregular heart rates, including tachycardia or bradycardia, causing symptoms such as palpitations and chest tightness.

[0004] Gene therapy can precisely target diseased genes and intervene at specific molecular targets, with a clear mechanism of action that helps reduce potential side effects. However, gene therapy is not yet widely used in the prevention and treatment of arrhythmias after myocardial ischemia-reperfusion. The main reason is that 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.

[0005] Transactive response DNA-binding protein 43 (TDP-43) is a DNA / RNA binding protein encoded by the TARDBP gene. In recent years, TDP-43 has been extensively studied in neurodegenerative diseases, and some researchers have begun to focus on its potential role in myocardial ischemia injury. Existing in vitro cell studies, by simulating myocardial ischemia injury through oxygen-glucose deprivation or hypoxia-reoxygenation treatment of HL-1 atrial myocytes, have found that TDP-43 expression is upregulated under ischemic stimulation, and knocking down TDP-43 or inhibiting its mitochondrial translocation can alleviate apoptosis and oxidative stress damage. Based on this, it is proposed that the TDP-43 knockdown strategy may have a cardioprotective effect.

[0006] However, the aforementioned studies were all limited to in vitro cultured cardiomyocyte models, with their observation endpoints being cellular / subcellular level damage indicators such as cell viability, apoptosis rate, and mitochondrial function. The occurrence of arrhythmias after myocardial ischemia-reperfusion involves myocardial electrical remodeling, altered expression and distribution of gap junction proteins, abnormal intercellular coupling function, and disruptions in overall cardiac electrical signal transduction pathways. These factors depend on the spatial arrangement of different cardiomyocyte types, intercellular synergy, and the intact electrical activity network of the heart, and cannot be observed and evaluated in in vitro single-cell models. Therefore, the results of the aforementioned in vitro cell studies are limited to the level of apoptosis regulation and do not address the changes in overall cardiac electrophysiological function after myocardial ischemia-reperfusion, nor do they link TDP-43 with the prevention and treatment of arrhythmias. Summary of the Invention

[0007] To address the lack of effective targets in the prevention and treatment of arrhythmias following myocardial ischemia-reperfusion, this invention provides the application of a TDP-43 overexpression vector in the preparation of drugs for the prevention and treatment of arrhythmias following myocardial ischemia-reperfusion.

[0008] The technical solution of the present invention:

[0009] Application of TDP-43 overexpression vector in the preparation of drugs for the prevention and treatment of arrhythmias after myocardial ischemia-reperfusion.

[0010] Furthermore, the TDP-43 overexpression vector is a recombinant vector containing the coding sequence of the TDP-43 gene, as shown in SEQ ID NO.1.

[0011] Furthermore, the recombinant vector is one of a plasmid vector, a bacteriophage vector, or a viral vector.

[0012] Furthermore, the viral vector is one of a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0013] Furthermore, the adeno-associated virus vector is the AAV9 serotype.

[0014] Furthermore, the arrhythmia following myocardial ischemia-reperfusion is a ventricular arrhythmia.

[0015] Furthermore, the drug for preventing and treating arrhythmias after myocardial ischemia-reperfusion has at least one of the following uses:

[0016] (1) Increases the expression level of TDP-43 protein in cardiac cardiomyocytes;

[0017] (2) Upregulate the mRNA and / or protein expression levels of gap junction protein Cx43 in myocardial tissue;

[0018] (3) Improve cardiac electrical conduction velocity after myocardial ischemia-reperfusion;

[0019] (4) Reduce the area of ​​myocardial infarction after myocardial ischemia-reperfusion;

[0020] (5) Reduce the rate of cardiomyocyte apoptosis after myocardial ischemia-reperfusion;

[0021] (6) Improves the ultrastructural damage of cardiomyocytes mitochondria after myocardial ischemia-reperfusion.

[0022] Furthermore, the drug for preventing and treating arrhythmias after myocardial ischemia-reperfusion is in injectable form.

[0023] Furthermore, the drug for preventing and treating arrhythmias after myocardial ischemia-reperfusion also contains pharmaceutically acceptable excipients.

[0024] The beneficial effects of this invention are:

[0025] This invention targets the TDP-43 gene, constructing a TDP-43 overexpression recombinant vector. In vivo experiments using a mouse model of myocardial ischemia-reperfusion significantly reduced the incidence of ventricular arrhythmias after myocardial ischemia-reperfusion, shortened arrhythmia induction time, increased cardiac electrical conduction velocity, and improved cardiac function. Mechanistic studies showed that TDP-43 overexpression upregulated the expression of the myocardial gap junction protein Cx43, reduced myocardial infarction area, decreased cardiomyocyte apoptosis rate, and improved mitochondrial ultrastructural damage. Mouse echocardiography showed a significant increase in left ventricular ejection fraction and short-axis shortening, a significant decrease in the incidence of programmed electrical stimulation-induced ventricular arrhythmias, and optical mapping showed a shortened cardiac activation time and increased conduction velocity. The above results indicate that gene therapy targeting TDP-43 can effectively inhibit the occurrence of arrhythmias after myocardial ischemia-reperfusion, providing a novel gene drug target and treatment strategy for the prevention and treatment of arrhythmias after myocardial ischemia-reperfusion, and has broad clinical application prospects. Attached Figure Description

[0026] Figure 1 The images shown are Western blot images of TDP-43 protein in the myocardial tissues of two groups of mice in Example 1. A is a protein electrophoresis band image, and B is a comparison of the relative expression levels of TDP-43 protein.

[0027] Figure 2 The images show the immunofluorescence staining detection of TDP-43 protein in the myocardial tissue of two groups of mice in Example 1. A is the immunofluorescence co-localization staining image of TDP-43 and myocardial cell markers, and B is the comparison of the relative fluorescence intensity of TDP-43.

[0028] Figure 3The images shown are Western blot images of TDP-43 protein in the myocardial tissues of two groups of mice in Example 2. A is a protein electrophoresis band image, and B is a comparison of the relative expression levels of TDP-43 protein.

[0029] Figure 4 M-mode echocardiography of the left ventricle in four groups of mice in Example 2;

[0030] Figure 5 This is a comparison chart of the core indicators of cardiac systolic function in four groups of mice in Example 2. A is a comparison chart of ejection fraction (EF) and B is a comparison chart of left ventricular fractional shortening (FS).

[0031] Figure 6 The following are comparison charts of left ventricular cavity diameter parameters in four groups of mice in Example 2: A is a comparison chart of left ventricular end-diastolic diameter (LVID;d), and B is a comparison chart of left ventricular end-systolic diameter (LVID;s).

[0032] Figure 7 The electrocardiograms of the four groups of mice in Example 3 are shown.

[0033] Figure 8 The following is a comparison chart of arrhythmia-related indicators in four groups of mice in Example 3: A is a comparison chart of arrhythmia induction time, and B is a comparison chart of arrhythmia incidence.

[0034] Figure 9 This is a thermal image of the spatial distribution of action potential time-course optical mapping of the epicardial membrane of the isolated mouse ventricle in Example 4.

[0035] Figure 10 The above are comparison charts of myocardial electrophysiological parameters in four groups of mice in Example 4. A is a comparison chart of myocardial electrical signal activation time and B is a comparison chart of myocardial electrical signal conduction velocity.

[0036] Figure 11 Transmission electron microscopy ultrastructure images of myocardial tissue from four groups of mice in Example 5;

[0037] Figure 12 The image shows a comparison of myocardial infarction area detected by TTC staining in the I / R group and the AAV9-cTnT-TDP-43+I / R group of mice in Example 6. A is a TTC staining image of a heart section, B is a comparison of the area of ​​the myocardial danger zone / left ventricular area, and C is a comparison of the area of ​​the infarction zone / danger zone.

[0038] Figure 13 The images shown are Western blot images of apoptosis-related proteins in the four groups of mice in Example 6. A is the electrophoresis band of Bax protein, B is the comparison of the relative expression levels of Bax protein, C is the electrophoresis band of Bcl-2 protein, and D is the comparison of the relative expression levels of Bcl-2 protein.

[0039] Figure 14The images show a comparison of TUNEL fluorescence staining detection of apoptosis in four groups of mouse myocardial tissues in Example 6. A is a TUNEL fluorescence staining image, and B is a comparison of the positive rate of TUNEL apoptosis in myocardial cells.

[0040] Figure 15 This is a comparison of the relative expression levels of Cx43 mRNA in the myocardium of four groups of mice in Example 7;

[0041] Figure 16 The images show the detection of Cx43 protein expression levels in the myocardium of four groups of mice in Example 7. A is an electrophoretic band image of Cx43 protein, and B is a comparison of the relative expression levels of Cx43 protein.

[0042] Figure 17 The images show the immunofluorescence localization staining of Cx43 in the myocardium of four groups of mice in Example 7. A is the immunofluorescence co-localization staining image of Cx43 and myocardial cell markers, and B is the comparison of the relative fluorescence intensity of Cx43.

[0043] Figure 18 Agarose gel electrophoresis image of TDP-43 myocardial condition knockout CKO-TDP-43 mice genotype identification in Example 8;

[0044] Figure 19 This is a comparison of the relative expression levels of TDP-43 mRNA in the myocardium of WT and CKO-TDP-43 mice in Example 8.

[0045] Figure 20 The image shows the TDP-43 protein expression levels in the myocardium of WT and CKO-TDP-43 mice in Example 8. A is the electrophoresis band of TDP-43 protein, and B is the comparison of the relative expression levels of TDP-43 protein.

[0046] Figure 21 The electrocardiograms of the two groups of mice in Example 8 are shown.

[0047] Figure 22 The above are comparison charts of arrhythmia-related indicators between the two groups of mice in Example 8. A is a comparison chart of arrhythmia induction time, and B is a comparison chart of arrhythmia incidence.

[0048] Figure 23 This is a comparison of the relative expression levels of Cx43 mRNA in the myocardium of the two groups of mice in Example 8;

[0049] Figure 24 The images show the expression levels of Cx43 protein in the myocardium of two groups of mice in Example 8. A is an electrophoretic band image of Cx43 protein, and B is a comparison of the relative expression levels of Cx43 protein.

[0050] Figure 25The images show the immunofluorescence localization staining of Cx43 in the myocardium of two groups of mice in Example 8. A is an image of Cx43 co-localization staining with myocardial cell markers using immunofluorescence, and B is a comparison of the relative fluorescence intensity of Cx43. Detailed Implementation

[0051] 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.

[0052] The experimental data in Examples 1-8 of this invention were statistically analyzed using GraphPad Prism 9.0 software. The data are expressed as mean ± standard error. One-way ANOVA was used for comparisons among multiple groups, and t-test was used for comparisons between two groups. P < 0.05 was used to indicate that the difference was statistically significant.

[0053] Example 1

[0054] In this embodiment, the expression level of TDP-43 in mouse myocardial ischemia-reperfusion tissue was detected by Western blot and immunofluorescence staining.

[0055] I. Experimental Materials

[0056] SPF-grade male C57BL / 6 mice, weighing approximately 25g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0057] Aphrodine anesthetic was purchased from Sigma (St. Louis, MO, USA).

[0058] Primary antibody: Rabbit anti-mouse TDP-43 antibody, purchased from Huaan Biotechnology, product number ET1703-74; Secondary antibody: IRDye®800CW Goat Anti-Rabbit, purchased from LI-COR, product number 926-32211; Fluorescent secondary antibody: Alexa Fluor®488 labeled goat anti-rabbit IgG fluorescent secondary antibody, purchased from abcam, product number AB150080.

[0059] OCT embedding agent and DAPI staining solution are both commercially available standard reagents.

[0060] II. Experimental Methods and Results

[0061] (I) Establishment of a mouse model of myocardial ischemia-reperfusion

[0062] Male C57BL / 6 mice weighing approximately 25g were used. After acclimatization, they were anesthetized by intraperitoneal injection of aphthylamine (0.22g / kg). The mice were fixed in a supine position on the operating table, shaved, and skin prepared. They were then intubated and connected to a ventilator for assisted ventilation. The muscles between the 2nd and 3rd ribs on the left chest were bluntly dissected to expose the heart. The left anterior descending coronary artery was ligated using 7 / 0 medical sutures. After ligation, the apex of the heart turned white, and ST segment elevation appeared on the electrocardiogram, indicating that the myocardial ischemia model was successfully established. After maintaining the ligation for 45 minutes, the sutures were released to restore coronary blood flow, and reperfusion was initiated. The skin on the left chest was sutured, and the mice were placed on a 37°C heating pad for resuscitation. After they were fully awake and in good condition, they were kept in a warm environment for another 24 hours to obtain a stable myocardial ischemia-reperfusion model mouse (I / R group).

[0063] In the sham group, mice were anesthetized and their hearts were opened. The sutures were passed under the left anterior descending branch but not ligated. The sutures were removed after 45 minutes and the mice were fed for another 24 hours.

[0064] (II) Western blot detection of TDP-43 protein expression in myocardial tissue

[0065] Twenty-four hours after reperfusion, myocardial tissue from the same location in both the I / R and Sham groups was used for Western blot experiments. 100 μl of pre-prepared cell lysis buffer was added to the extracted tissue, followed by sonication and centrifugation at 13500 rpm for 15 minutes at 4°C. After centrifugation, the supernatant was transferred to a new 1.5 ml EP tube; the supernatant was the extracted total protein. First, the prepared SDS-PAGE gel was placed in the electrophoresis tank, and electrophoresis buffer was added to the appropriate mark. Protein samples were loaded in sequence according to experimental requirements, 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. The gel was removed using a gel release plate and placed in the following order: white sponge - filter paper - NC membrane - gel - filter paper - black sponge, gently removing air bubbles. The transfer tank was placed on ice to maintain a low temperature, and the membrane was transferred at a constant current of 300 mA for 40 minutes. The NC membrane was placed in a pre-prepared blocking buffer containing 10% skim milk and blocked on a shaker at room temperature for 1.5 hours. The target band was cut from the blocked NC membrane according to the molecular weight of the target protein and placed separately in dilution buffers for TDP-43 primary antibody and α-Tubulin internal control antibody, and incubated overnight at 4°C in a resealable bag. The NC membrane was then removed and washed four times with PBST for 7 minutes each time, followed by incubation with diluted secondary antibody solution at room temperature for 50 minutes. ECL chemiluminescence imaging was then performed for analysis. Using α-Tubulin (a-actin) as an internal control, the gray values ​​of the bands were quantitatively analyzed using ImageJ software. The relative expression level of TDP-43 protein was expressed as the ratio of the gray values ​​of TDP-43 to α-Tubulin.

[0066] The results are as follows Figure 1 As shown, compared with the Sham group, the expression level of TDP-43 protein in the myocardial tissue of I / R group mice was significantly increased.

[0067] (III) Immunofluorescence staining to detect the expression and distribution of TDP-43 in myocardial tissue

[0068] Myocardial tissue from I / R group mice and Sham group mice was embedded in OCT and then flash-frozen in liquid nitrogen. Sections were prepared using a cryostat, fixed with pre-chilled acetone for 7 minutes, and washed three times (5 minutes each) on a PBS shaker. For the breakthrough buffer, 30 μl of Triton was dissolved in 10 ml of PBS. An appropriate amount of breakthrough buffer was added to each well to cover the cell fragments, and the cells were incubated at room temperature for 1 hour. The cells were blocked with undiluted goat serum and incubated at room temperature for 30 minutes. After blocking, the blocking solution was discarded, and the cells were washed three times (5 minutes each) with PBST. The primary antibody was diluted according to the manufacturer's instructions and incubated overnight at 4°C. The primary antibody was washed three times (5 minutes each) with PBST. For the fluorescent secondary antibody, 50 μl of the prepared secondary antibody was added to each well in the dark and incubated at 37°C for 1.5 hours in the dark. The secondary antibody was washed three times (5 minutes each) with PBST. Prepare DAPI according to the specified ratio, avoiding light. Add 50 μl of the prepared DAPI to each well, incubate at room temperature for 15 minutes, discard the DAPI staining solution, and wash the cells with PBST for 5 minutes each time, for a total of 3 times. Under light-protected conditions, place the treated cells under a laser confocal microscope for imaging.

[0069] The results are as follows Figure 2 As shown, compared with the Sham group, the TDP-43 fluorescence signal in the myocardial tissue of the I / R group mice was significantly enhanced. Quantitative fluorescence intensity analysis showed that the TDP-43 fluorescence intensity in the I / R group was significantly higher than that in the Sham group, further confirming that TDP-43 expression was upregulated after myocardial ischemia-reperfusion.

[0070] Example 2

[0071] In this embodiment, a recombinant adeno-associated virus vector AAV9-TDP-43 overexpressing TDP-43 was constructed and injected into mice via the tail vein. The effect of TDP-43 overexpression on cardiac function in mice with myocardial ischemia-reperfusion was detected by echocardiography.

[0072] I. Experimental Materials

[0073] SPF-grade male C57BL / 6 mice, weighing approximately 25g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0074] The TDP-43 gene overexpression plasmid and adeno-associated virus were constructed and packaged by Suzhou Gemma Gene Co., Ltd.: using the AAV9 plasmid as a vector, the full-length murine TDP-43 nucleotide sequence and the enhanced green fluorescent protein (EGFP) sequence as shown in SEQ ID NO.2 were inserted, and the AAV9-TDP-43 overexpression recombinant plasmid was constructed using CMV as a promoter; the negative control plasmid was constructed and packaged by Suzhou Gemma Gene Co., Ltd.: only irrelevant blank sequences were inserted, and the remaining vector elements were completely identical to those of the overexpression plasmid.

[0075] Since the animal model used in this embodiment is a mouse, the overexpression intervention of the mouse TDP-43 gene can avoid the immunogenicity or receptor affinity bias that may occur in mice due to species differences in human TDP-43, thus more accurately reflecting the true efficacy of TDP-43 in target organs. Although the efficacy verification in this embodiment uses the mouse TDP-43 gene, this technical solution has cross-species universality. According to sequence alignment analysis of public databases such as UniProt and NCBI, human and mouse TDP-43 proteins are clearly orthologous genes with highly conserved amino acid sequences (the identity between human TDP-43 and mouse TDP-43 is as high as 96%), and its main functional mechanisms such as nuclear localization, RNA binding, and mitochondrial translocation are highly conserved across species. Therefore, the overexpression intervention effect and antiarrhythmic effect verified on mouse TDP-43 can be reasonably extrapolated to the application scenario of human TDP-43. This inference is in line with the conventional research paradigm of extrapolating cross-species efficacy based on evolutionary conservation in this field, and provides reliable experimental evidence for clinical translation.

[0076] Recombinant plasmids overexpressing the TDP-43 gene or negative control plasmids were co-transfected with AAV packaging helper plasmids into HEK293 cells. Recombinant adeno-associated virus (AAV) was then assembled using an intracellular expression system. Cell culture supernatants were collected, concentrated, purified, and impurities were removed to obtain high-purity mature AAV9-TDP-43 and AAV9-NC viral particles, respectively. All constructed plasmids underwent full-length sequencing identification, and sequence alignment results showed no mutations or deletions, indicating accurate and reliable gene sequences.

[0077] Afodin anesthetic was purchased from Sigma (St. Louis, MO, USA), and the ultrasound coupling agent was a commercially available standard reagent.

[0078] II. Experimental Methods and Results

[0079] (a) Animal grouping and AAV9 virus injection

[0080] Male C57BL / 6 mice were acclimatized and then randomly divided into four groups:

[0081] Sham group: Male C57BL / 6 mice were used. No virus was injected. The thorax was opened according to the procedure in Example 1. The left anterior descending coronary artery was not ligated. The other surgical procedures and feeding conditions were the same as those in the model group.

[0082] Mice with myocardial ischemia-reperfusion injury (I / R group): No virus was injected, and the myocardial ischemia-reperfusion injury model was constructed strictly according to the method in Example 1;

[0083] AAV9-TDP-43+I / R group: Each mouse was injected via tail vein with 150 μL of AAV9-TDP-43 virus diluent, containing 2 × 10⁻⁶ cells / mL. 11 One genome particle (GC) was injected and the patient was fed for 4 weeks. Then, a myocardial ischemia-reperfusion injury model was constructed strictly according to the method in Example 1.

[0084] AAV9-NC+I / R group: Each mouse was injected via tail vein with 150 μL of AAV9-NC virus diluent, containing 2 × 10⁻⁶ viruses. 11 A genome particle (GC) was injected and the patient was fed for 4 weeks. Then, a myocardial ischemia-reperfusion injury model was constructed strictly according to the method in Example 1.

[0085] (II) Western blot detection of TDP-43 protein expression in myocardial tissue

[0086] Four weeks after the viral injection, mice were routinely fed. Myocardial tissues from mice injected with AAV9-TDP-43 virus and AAV9-NC virus via tail vein were collected. Total protein was extracted and Western blot analysis was performed according to the method in Example 1. α-Tubulin was used as an internal reference protein to correct for differences in sample loading, and the protein expression level of TDP-43 was quantitatively analyzed.

[0087] The results are as follows Figure 3 As shown, compared with mice injected with AAV9-NC virus, mice injected with AAV9-TDP-43 virus showed significantly increased TDP-43 protein expression in myocardial tissue, with statistically significant differences. This confirms that AAV9-TDP-43 virus can stably achieve TDP-43 protein overexpression in mouse myocardial tissue, and the viral intervention model was successfully constructed.

[0088] (III) Construction of a myocardial ischemia-reperfusion model and echocardiographic detection

[0089] Twenty-four hours after reperfusion, echocardiography was performed on mice in each group. After weighing, mice were anesthetized by intraperitoneal injection of afodin, fixed in a supine position on a 37°C constant-temperature operating platform, their limbs were secured with tape, and after hair removal, ultrasound coupling agent was applied to their chests. M-mode ultrasound images of the left ventricle in the long and short axes were acquired using a VINNO 6 high-resolution imaging system. The left ventricular end-diastolic diameter (LVID;d) and left ventricular end-systolic diameter (LVID;s) were measured, and the ejection fraction (EF%) and left ventricular short-axis shortening rate (FS%) were calculated to assess cardiac function.

[0090] Echocardiograms of the four groups of mice are as follows: Figure 4 As shown in the figure, the comparison between ejection fraction (EF) and left ventricular fractional shortening (FS) is as follows. Figure 5As shown in the figure, the comparison between the left ventricular end-diastolic diameter (LVID) and the end-systolic diameter (LVID) is as follows. Figure 6 As shown, compared with the Sham group, the I / R group model mice showed significantly reduced left ventricular EF% and FS%, and significantly increased LVID;d and LVID;s, indicating that ischemia-reperfusion injury impaired myocardial contractile function, increased ventricular diameter, and ventricular dilatation lesions.

[0091] Compared with the AAV9-NC+I / R group, the AAV9-TDP-43+I / R group showed significantly increased EF% and FS%, and significantly decreased LVID;d and LVID;s, indicating that overexpression of TDP-43 can repair myocardial contractile function and alleviate ventricular structural abnormalities caused by ischemia-reperfusion. There were no statistically significant differences in any of the indicators between the AAV9-NC+I / R group and the I / R model group.

[0092] This embodiment demonstrates that TDP-43 overexpression can significantly improve cardiac function damage in mice caused by myocardial ischemia-reperfusion.

[0093] Example 3

[0094] This embodiment uses programmed electrical stimulation to detect the effect of TDP-43 overexpression on ventricular arrhythmias in mice with myocardial ischemia-reperfusion.

[0095] I. Experimental Materials

[0096] In this example, the mice, TDP-43 gene overexpression plasmid, negative control plasmid, and adeno-associated virus were the same as in Example 2.

[0097] II. Experimental Methods and Results

[0098] In this embodiment, based on the mouse models with different groups in Example 2, programmed electrical stimulation was performed on each group of mice to detect the incidence of ventricular arrhythmias after I / R.

[0099] The specific methods of programmed electrical stimulation are as follows:

[0100] Twenty-four hours after reperfusion, the mice were anesthetized and 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 through this incision into the right ventricle of the mouse. The stimulation pattern consisted of 10 consecutive electrical pulses (S1) with a coupling interval of 80 ms, followed by two additional stimuli (S2 and S3), with the coupling interval decreasing by -2 ms from 80 ms. The occurrence of arrhythmias was recorded at 3.5 V, 5 V, and 8 V. The stimulation was repeated twice to check the reproducibility of the experiment.

[0101] The electrocardiograms of the four groups of mice are as follows: Figure 7As shown in the figure, the comparison chart of arrhythmia-related indicators is as follows: Figure 8 As shown, compared with the Sham group, the incidence of ventricular arrhythmias in the I / R model group mice was significantly increased, and the induction time of ventricular arrhythmias was significantly prolonged. Compared with the AAV9-NC+I / R group, the incidence of ventricular arrhythmias in the AAV9-TDP-43+I / R group mice was significantly decreased, and the induction time was significantly shortened, while there was no statistically significant difference between the AAV9-NC+I / R group and the I / R model group.

[0102] This embodiment demonstrates that TDP-43 overexpression can significantly reduce the occurrence of ventricular arrhythmias after myocardial ischemia-reperfusion.

[0103] Example 4

[0104] In this embodiment, the effect of TDP-43 overexpression on cardiac electrical conduction function in mice with myocardial ischemia-reperfusion injury was detected by electrical mapping technology.

[0105] I. Experimental Materials

[0106] In this example, the mice, TDP-43 gene overexpression plasmid, negative control plasmid, and adeno-associated virus were the same as in Example 2.

[0107] II. Experimental Methods and Results

[0108] Impaired conduction of myocardial action potentials can lead to arrhythmias by forming slow conduction return pathways. In this example, based on mouse models with different groups from Example 2, electrical mapping technology was used to detect changes in cardiac activation duration and conduction velocity in each group of mice.

[0109] The specific method for electrical mapping is as follows:

[0110] Twenty-four hours after reperfusion, the mice were anesthetized and euthanized. The thoracic cavity was quickly opened, and the heart was immediately removed and placed in the perfusion fluid. The aorta was attached to the perfusion needle, and perfusion fluid was injected to drain as much blood as possible from the heart. At 37°C, the perfusion needle was connected to the Langendorff perfusion device. After the drip rate stabilized and the heart was free of blood, a flexible multi-electrode patch was attached to the epicardium. Baseline electrophysiological mapping was performed, and conduction and repolarization mapping was reconstructed offline to record the overall electrical signal changes of the heart.

[0111] The spatial distribution heatmaps of action potential time-course optical mapping of four groups of isolated mouse ventricular epicardial membranes are shown below. Figure 9As shown, the myocardial electrical signal distribution in the Sham group was uniform and regular, with a smooth color transition, indicating normal myocardial electrical conduction. Large areas in the I / R group and the AAV9-NC+I / R group appeared dark blue, indicating disordered and uneven distribution of electrical activity, suggesting that ischemia-reperfusion injury disrupted the normal myocardial electrical signal diffusion pattern. The AAV9-TDP-43+I / R group showed significantly improved uniformity of electrical signal distribution and a substantial reduction in abnormally low-activity areas, indicating that TDP-43 overexpression can repair the disordered myocardial electrical distribution caused by ischemic injury.

[0112] Comparison of myocardial electrophysiological parameters as shown in the figure Figure 10 As shown, compared with the Sham group, the conduction time of the heart in the I / R group mice was prolonged when traveling the same distance, indicating that the cardiac activation time was significantly slowed in the I / R group mice. AAV9-TDP-43 treatment can significantly shorten the myocardial activation time and restore the normal electrical activation rhythm of the myocardium. Furthermore, compared with the Sham group, the conduction velocity of the I / R group mice was slowed, which delays the time for cells to return to an excitable state, increases the risk of cardiac conduction block, and easily induces reentry electrical activity and arrhythmias. AAV9-TDP-43 overexpression can significantly restore myocardial conduction velocity, while the unloaded control group showed no repair effect.

[0113] This embodiment demonstrates that TDP-43 overexpression in mouse hearts can improve electrophysiological abnormalities such as myocardial conduction block and delayed electrical activity caused by ischemia-reperfusion injury.

[0114] Example 5

[0115] In this embodiment, the effect of TDP-43 overexpression on the ultrastructure of cardiomyocytes in mice with myocardial ischemia-reperfusion was observed by transmission electron microscopy.

[0116] I. Experimental Materials

[0117] In this example, the mice, TDP-43 gene overexpression plasmid, negative control plasmid, and adeno-associated virus were the same as in Example 2.

[0118] II. Experimental Methods and Results

[0119] In this embodiment, based on the mouse models with different groups in Example 2, the left ventricular myocardial tissue of each group of mice was taken for transmission electron microscopy observation.

[0120] The sample preparation and observation methods for transmission electron microscopy are as follows:

[0121] Twenty-four hours after reperfusion, left ventricular myocardial tissue was harvested from each group of mice and rapidly trimmed to 1 mm. 3Small tissue blocks were immediately immersed in pre-cooled 2.5% glutaraldehyde fixative and fixed at 4°C. After thorough rinsing with 0.1 mol / L PBS, the blocks were fixed with 1% osmium tetroxide at room temperature, dehydrated with a gradient of ethanol and acetone, and embedded in Epon 812 epoxy resin. The embedded blocks were then prepared into 70 nm ultrathin sections using an ultramicrotome. After double staining with uranium acetate and lead citrate, the sections were observed under a transmission electron microscope, and ultrastructural images of the myocardium were acquired.

[0122] Ultrastructure images such as Figure 11 As shown, in Sham mice, myofibrils in cardiac tissue were arranged regularly, and sarcomere structures were intact and clear (blue arrows). Mitochondria were plump, with dense and continuous cristae without swelling or damage (green arrows). Cytoplasm showed no vacuolation or edema, and the intercalated disc structure was dense and continuous. Compared with the Sham group, I / R mice and AAV9-NC+I / R empty vector control mice showed typical ultrapathological changes of myocardial injury, including myofibril dissolution and breakage, disordered myofibril arrangement, widespread mitochondrial swelling, cristae dissolution and loss, significant expansion and vacuolation of endoplasmic reticulum (red arrows), accompanied by intercellular and perinuclear edema (purple arrows) and interruption of intercalated disc structure. In contrast, the ultrastructural damage of cardiac tissue in AAV9-TDP-43 overexpression mice was significantly alleviated, myofibrils and sarcomeres were arranged more regularly, mitochondrial swelling was significantly reduced, cristae structure integrity was improved, intercalated disc continuity was preserved (yellow arrows), and cytoplasmic vacuolation and cellular edema were significantly reduced.

[0123] This embodiment demonstrates that TDP-43 overexpression can effectively improve I / R-induced ultrastructural damage to cardiomyocyte organelles, maintain the integrity of key structures such as the cardiomyocyte cytoskeleton, mitochondria, and intercalated discs, and exert a cardioprotective effect.

[0124] Example 6

[0125] In this embodiment, the effects of TDP-43 overexpression on myocardial infarction area and cardiomyocyte apoptosis in mice with myocardial ischemia-reperfusion were detected by TTC staining, Western blot, and TUNEL immunofluorescence staining.

[0126] I. Experimental Materials

[0127] The recombinant adeno-associated virus used in this example was AAV9-cTnT-TDP-43 (specifically expressed in cardiomyocytes by the cardiac troponin T promoter), constructed and packaged by Suzhou Gemma Gene Co., Ltd., and stored at -80℃ for later use. The remaining mice, reagents, and materials were the same as in Example 2.

[0128] TTC staining solution, Bax antibody, Bcl-2 antibody, α-actinin antibody, and TUNEL apoptosis detection kit are all commercially available products.

[0129] II. Experimental Methods and Results

[0130] (a) Animal grouping and AAV9 virus injection

[0131] Sham group: Male C57BL / 6 mice were used. No virus was injected. The thorax was opened according to the procedure in Example 1. The left anterior descending coronary artery was not ligated. The other surgical procedures and feeding conditions were the same as those in the model group.

[0132] AAV9-cTnT-TDP-43 group: Each mouse was injected via tail vein with 150 μl of AAV9-cTnT-TDP-43 virus diluent, containing 2 × 10⁻⁶ cells / mL. 11 One genome particle (GC) was injected and the patient was fed for 4 weeks without constructing a myocardial ischemia-reperfusion injury model.

[0133] Mice with myocardial ischemia-reperfusion injury (I / R group): No virus was injected, and the myocardial ischemia-reperfusion injury model was constructed strictly according to the method in Example 1;

[0134] AAV9-cTnT-TDP-43+I / R group: Each mouse was injected via tail vein with 150 μL of AAV9-cTnT-TDP-43 virus diluent, containing 2 × 10⁻⁶ cells / mL. 11 One genome particle (GC) was injected and the patient was fed for 4 weeks. Then, a myocardial ischemia-reperfusion injury model was constructed strictly according to the method in Example 1.

[0135] (II) TTC staining to detect myocardial infarction area

[0136] Twenty-four hours after reperfusion, the hearts of mice in the I / R group and the AAV9-cTnT-TDP-43+I / R group were harvested, frozen at -20°C, and cut into 1-2 mm thick sections. These sections were then incubated in 1% TTC staining solution at 37°C in the dark for 15-20 minutes. After staining, normal myocardium appeared red, while infarcted myocardium appeared white. ImageJ software was used to calculate the percentage of the infarct area relative to the ischemic risk area (AAR) after image acquisition.

[0137] TTC staining results are as follows Figure 12 As shown, compared with the I / R group, the proportion of myocardial infarction area to ischemic risk area was significantly reduced in the AAV9-cTnT-TDP-43+I / R group of mice, indicating that TDP-43 overexpression can significantly reduce the infarct size of myocardial ischemia-reperfusion injury.

[0138] (III) Western blot detection of apoptosis-related protein expression in myocardial tissue

[0139] Twenty-four hours after reperfusion, left ventricular myocardial tissue was collected from each group of mice, total protein was extracted, protein was quantified by BCA method, separated by SDS-PAGE electrophoresis and transferred to NC membrane, blocked with 5% skim milk at room temperature for 1.5 hours, Bax antibody and Bcl-2 antibody were added respectively, incubated at 4°C overnight, washed with PBST, added HRP-labeled secondary antibody and incubated at room temperature for 50 minutes, ECL chemiluminescence imaging, α-Tubulin as internal control, and the gray value of the bands was quantitatively analyzed using ImageJ software.

[0140] Western blot results are as follows Figure 13 As shown, there was no statistically significant difference in the relative expression level of Bax between the Sham group and the AAV9-cTnT-TDP-43 group, indicating that high expression of TDP-43 in the myocardium alone does not affect the basal apoptosis protein levels in normal myocardium. Compared to the Sham group, the Bax protein expression level in the I / R group was significantly increased, suggesting that ischemia-reperfusion injury induces a large amount of pro-apoptotic protein Bax expression, initiating the cardiomyocyte apoptosis program. Compared to the I / R group, the Bax expression level in the AAV9-cTnT-TDP-43+I / R group was significantly downregulated, demonstrating that myocardial-specific overexpression of TDP-43 can inhibit the upregulation of pro-apoptotic proteins induced by ischemia injury and weaken pro-apoptotic signals.

[0141] There was no significant difference in Bcl-2 expression between the Sham group and the AAV9-cTnT-TDP-43 group, and TDP-43 itself does not alter the baseline expression of Bcl-2 in normal myocardium. After I / R injury, myocardial Bcl-2 protein levels decreased significantly, the body's anti-apoptotic ability was significantly suppressed, and the myocardial protective mechanism against apoptosis was impaired. After intervention with myocardial-targeted TDP-43, Bcl-2 expression in the AAV9-cTnT-TDP-43+I / R group significantly increased compared to the I / R group, indicating that TDP-43 overexpression can restore the level of anti-apoptotic proteins in damaged myocardium.

[0142] (iv) TUNEL immunofluorescence staining for cardiomyocyte apoptosis

[0143] Twenty-four hours after reperfusion, frozen sections of left ventricular myocardial tissue were prepared from mice in each group. Apoptotic cell nuclei were labeled with TUNEL staining (red), while the cardiomyocyte cytoskeleton was labeled with α-actinin (green) and cell nuclei were labeled with DAPI (blue). Images were acquired using laser confocal microscopy, and the TUNEL-positive cell rate (number of TUNEL-positive cells / total number of cells × 100%) was calculated.

[0144] TUNEL staining results are as follows: Figure 14As shown, almost no red TUNEL positive apoptosis signal was observed in the myocardial tissue field of the Sham group and the AAV9-cTnT-TDP-43 group, and the myocardial fibers were arranged in a regular and orderly manner. This indicates that under physiological conditions, the myocardium-specific overexpression of TDP-43 alone will not induce apoptosis in normal myocardial cells. TDP-43 itself has no cytotoxicity to healthy myocardium and will not disrupt the normal survival homeostasis of myocardium.

[0145] Compared to the Sham group, the I / R group showed a large number of red TUNEL punctate fluorescent signals in the sections. Under magnification, apoptotic cells were widely distributed between myocardial fibers, and the positive rate of myocardial TUNEL was significantly increased. This indicates that ischemia-reperfusion injury causes a large number of myocardial DNA breaks, significantly inducing myocardial cell apoptosis, which is an important mechanism of myocardial injury caused by I / R.

[0146] Compared to the I / R group, the AAV9-cTnT-TDP-43+I / R group showed a significant reduction in the number of red apoptotic fluorescence foci and a marked improvement in apoptotic lesions in myocardial tissue; its TUNEL positivity rate was also significantly lower than that of the I / R group. This demonstrates that pre-expression of TDP-43 via a myocardial targeting vector can effectively inhibit ischemia-reperfusion-induced cardiomyocyte apoptosis and significantly reduce the proportion of apoptotic cells.

[0147] This embodiment demonstrates that TDP-43 overexpression can significantly reduce the infarct size after myocardial ischemia-reperfusion and inhibit cardiomyocyte apoptosis by regulating Bax / Bcl-2 expression.

[0148] Example 7

[0149] In this embodiment, the effect of TDP-43 overexpression on the expression of gap junction protein Cx43 in myocardial tissue of mice with myocardial ischemia-reperfusion was detected by qRT-PCR, Western blot and immunofluorescence staining.

[0150] I. Experimental Materials

[0151] In this example, the mice, TDP-43 gene overexpression plasmid, negative control plasmid, and adeno-associated virus were the same as in Example 2. qRT-PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the Cx43 antibody was purchased from ProteinTech (product number 26980-1-AP); the secondary antibody was IRDye® 800CW Goat Anti-Rabbit, purchased from LI-COR (product number 926-32211); the fluorescent secondary antibody was purchased from Abcam (product number AB150080); and the RNA extraction kit and reverse transcription kit were commercially available products.

[0152] II. Experimental Methods and Results

[0153] Based on the mouse models with different groups in Example 2, this embodiment uses left ventricular myocardial tissue from each group of mice and detects the changes in Cx43 mRNA expression level, protein expression level and fluorescence intensity by qRT-PCR, Western blot and immunofluorescence staining.

[0154] (a) qRT-PCR detection of Cx43 mRNA expression in myocardial tissue

[0155] Twenty-four hours after reperfusion, myocardial tissue from the same location was taken from mice in each group. Total RNA was extracted using the Trizol method, reverse transcribed into cDNA, and then the expression level of Cx43 mRNA was detected by real-time fluorescence quantitative PCR using SYBR Green method with β-actin as an internal control.

[0156] The Cx43 primer sequence is:

[0157] SEQ ID NO.3 (Mouse-cx43-F): 5'-ACTTCAATGGCTGCTCCTCACC-3',

[0158] SEQ ID NO.4 (Mouse-cx43-R): 5'-GCTCGCTGGCTTGCTTGTTG-3';

[0159] The β-actin primer sequence is as follows:

[0160] SEQ ID NO.5 (Mouse β-actin-F):

[0161] 5'-ATGCCACAGGATTCCATACCCAAGA-3',

[0162] SEQ ID NO.6 (Mouse β-actin-R):

[0163] 5'-CTCTAGACTTCGAGCAGGAGATGG-3'.

[0164] Use 2 -ΔΔCt The relative expression level of Cx43 mRNA was calculated using this method.

[0165] qRT-PCR results are as follows Figure 15 As shown, compared with the Sham group, the expression level of Cx43 mRNA in the myocardial tissue of mice in the I / R group was significantly reduced. Compared with the AAV9-NC+I / R group, the expression level of Cx43 mRNA in the AAV9-TDP-43+I / R group was significantly increased, while there was no statistically significant difference between the AAV9-NC+I / R group and the I / R group.

[0166] (II) Western blot detection of Cx43 protein expression in myocardial tissue

[0167] Twenty-four hours after reperfusion, myocardial tissue from the same location in each group of mice was collected, total protein was extracted, protein was quantified by BCA method, separated by SDS-PAGE electrophoresis and transferred to NC membrane, blocked with 5% skim milk at room temperature for 1.5 hours, Cx43 antibody was added and incubated overnight at 4°C, washed with PBST and incubated with HRP-labeled secondary antibody at room temperature for 50 minutes, ECL chemiluminescence imaging was performed, α-Tubulin was used as an internal control, and the gray values ​​of the bands were quantitatively analyzed using ImageJ software.

[0168] Western blot results are as follows Figure 16 As shown, compared with the Sham group, the expression level of Cx43 protein in the myocardial tissue of mice in the I / R group was significantly reduced. Compared with the AAV9-NC+I / R group, the expression level of Cx43 protein in the AAV9-TDP-43+I / R group was significantly increased, while there was no statistically significant difference between the AAV9-NC+I / R group and the I / R model group.

[0169] (III) Immunofluorescence staining to detect the expression and distribution of Cx43 in myocardial tissue

[0170] Twenty-four hours after reperfusion, frozen sections of left ventricular myocardial tissue from each group of mice were prepared. Cx43 antibody was incubated overnight at 4°C, and fluorescent secondary antibody was incubated at 37°C in the dark for 1.5 hours. The nuclei were stained with DAPI, and images were acquired using a laser confocal microscope. The fluorescence intensity of Cx43 was quantitatively analyzed using ImageJ software.

[0171] Immunofluorescence staining results as follows Figure 17 As shown, compared with the Sham group, the Cx43 fluorescence signal in the myocardial tissue of mice in the I / R group was significantly weakened. Compared with the AAV9-NC+I / R group, the Cx43 fluorescence intensity in the AAV9-TDP-43+I / R group was significantly enhanced, while there was no statistically significant difference between the AAV9-NC+I / R group and the I / R model group.

[0172] This embodiment demonstrates that TDP-43 overexpression can significantly upregulate the mRNA and protein expression levels of gap junction protein Cx43 in myocardial tissue after myocardial ischemia-reperfusion, maintain the integrity of gap junction structure, improve myocardial electrical coupling damage caused by myocardial ischemia-reperfusion, correct the pathological conditions of slow ventricular conduction and reentry, and ultimately alleviate I / R-induced cardiac electrical conduction disorders and reduce susceptibility to arrhythmias. It jointly demonstrates the protective effect of TDP-43 on the electrophysiology of ischemic myocardium at both molecular and functional levels.

[0173] Example 8

[0174] This embodiment uses the construction of TDP-43 myocardial specific conditional knockout mice (CKO-TDP-43) to reversely verify the regulatory effect of TDP-43 on ventricular arrhythmias and Cx43 expression after myocardial ischemia-reperfusion.

[0175] I. Experimental Materials

[0176] The TDP-43 gene knockout transgenic mouse CKO-TDP-43 was constructed by Cyagen Biosciences using CRISPR / Cas9 gene editing technology.

[0177] The nucleotide sequences of the PCR identification primers are as follows:

[0178] SEQ ID NO.7 (F1): 5'-CAAGCCTGACAATCCTATTTGGAA-3',

[0179] SEQ ID NO. 8 (R1): 5'-GGATATTTGTAAGCAGGTTCACACC-3'.

[0180] The TDP-43 antibody, Cx43 antibody, fluorescent secondary antibody, RNA extraction kit, reverse transcription kit, SYBR Green real-time PCR kit, RIPA lysis buffer, BCA protein quantification kit, OCT embedding agent, and DAPI staining solution were all the same as in Example 7. Other conventional reagents were the same as in the aforementioned examples.

[0181] II. Experimental Methods and Results

[0182] (I) Genotyping of CKO-TDP-43 mice

[0183] Genomic DNA was extracted from the tail tips of CKO-TDP-43 mice and littermate WT mice, and genotypes were identified by PCR amplification and agarose gel electrophoresis.

[0184] Genotyping results as follows Figure 18 As shown, the CKO-TDP-43 mouse genome can specifically amplify the Cre recombinase gene band and the TDP-43 knockout allele fragment. Wild-type mice only show the wild-type allele band. The pure water blank control group has no specific amplification products, excluding exogenous nucleic acid contamination. The gene modification was confirmed to be successful at the genomic level.

[0185] (II) Verification of TDP-43 knockout efficiency in CKO-TDP-43 mouse myocardial tissue

[0186] Total RNA was extracted from the myocardial tissues of CKO-TDP-43 and WT mice, reverse transcribed into cDNA, and then subjected to qRT-PCR with β-actin as an internal control to detect the expression level of TDP-43 mRNA. Total protein was also extracted from the myocardial tissues, and the expression level of TDP-43 protein was detected by Western blot, with [internal control name] as the internal control. ECL chemiluminescence imaging was used, and ImageJ software was used for quantitative analysis of the band gray values.

[0187] qRT-PCR results are as follows Figure 19 As shown, compared with WT mice, the expression level of TDP-43 mRNA in the myocardial tissue of CKO-TDP-43 mice was significantly reduced. Western blot results are as follows... Figure 20 As shown, compared with WT mice, the expression level of TDP-43 protein in the myocardial tissue of CKO-TDP-43 mice was significantly reduced. These results confirm the successful construction of CKO-TDP-43 mice and the effective knockout of TDP-43 in myocardial tissue.

[0188] (III) Effects of TDP-43 knockout on ventricular arrhythmias after myocardial ischemia-reperfusion

[0189] CKO-TDP-43 mice and WT mice were used to establish an I / R model (ligation for 45 minutes / reperfusion for 24 hours) according to the method in Example 1. After 24 hours of reperfusion, the incidence and induction duration of ventricular arrhythmias were detected using the programmed electrical stimulation method in Example 3.

[0190] Results of programmed electrical stimulation such as Figure 21 and Figure 22 As shown, compared with the WT group, the incidence of programmed electrical stimulation-induced ventricular arrhythmias was significantly increased and the induction time of ventricular arrhythmias was significantly prolonged in TDP-43 knockout mice. TDP-43 knockout promoted susceptibility to and persistence of ventricular arrhythmias.

[0191] (iv) Effects of TDP-43 knockout on Cx43 expression in myocardial tissue

[0192] Myocardial tissues from CKO-TDP-43 and WT mice were collected. The expression level of Cx43 mRNA was detected by qRT-PCR, the expression level of Cx43 protein was detected by Western blot, and the fluorescence intensity and distribution of Cx43 were detected by immunofluorescence staining. The specific methods were the same as in Example 7.

[0193] qRT-PCR results are as follows Figure 23 As shown, compared with WT mice, the expression level of Cx43 mRNA in the myocardial tissue of CKO-TDP-43 mice was significantly reduced. Western blot results are as follows... Figure 24As shown, compared with WT mice, the expression level of Cx43 protein in the myocardial tissue of CKO-TDP-43 mice was significantly reduced. Immunofluorescence staining results are shown below. Figure 25 As shown, compared with WT mice, the fluorescence intensity of Cx43 in the myocardial tissue of CKO-TDP-43 mice was significantly reduced. Inhibition of Cx43 expression directly disrupts the electrical signal coupling between cardiomyocytes, causing a decrease in the myocardial excitation conduction rate, which in turn promotes the occurrence of arrhythmias.

[0194] This embodiment confirms that TDP-43 knockout can significantly downregulate the expression of Cx43 in myocardial tissue and significantly increase the incidence and prolong the induction time of ventricular arrhythmias after myocardial ischemia-reperfusion. This provides positive and negative evidence for the protective effect of TDP-43 overexpression in Examples 2-7, further confirming that TDP-43 exerts its antiarrhythmic effect by upregulating Cx43 expression and maintaining gap junction function.

Claims

1. Application of TDP-43 overexpression vector in the preparation of drugs for the prevention and treatment of arrhythmias after myocardial ischemia-reperfusion.

2. The application according to claim 1, characterized in that, The TDP-43 overexpression vector is a recombinant vector containing the coding sequence of the TDP-43 gene, as shown in SEQ ID NO.

1.

3. The application according to claim 2, characterized in that, The recombinant vector is a viral vector.

4. The application according to claim 3, characterized in that, The viral vector is an adeno-associated virus vector.

5. The application according to claim 4, characterized in that, The adeno-associated virus vector is the AAV9 serotype.

6. The application according to any one of claims 1-5, characterized in that, The arrhythmia following myocardial ischemia-reperfusion is a ventricular arrhythmia.

7. The application according to claim 6, characterized in that, The drug for preventing and treating arrhythmias after myocardial ischemia-reperfusion has at least one of the following uses: (1) Increases the expression level of TDP-43 protein in cardiac cardiomyocytes; (2) Upregulate the mRNA and / or protein expression levels of gap junction protein Cx43 in myocardial tissue; (3) Improve cardiac electrical conduction velocity after myocardial ischemia-reperfusion; (4) Reduce the area of ​​myocardial infarction after myocardial ischemia-reperfusion; (5) Reduce the rate of cardiomyocyte apoptosis after myocardial ischemia-reperfusion; (6) Improves the ultrastructural damage of cardiomyocytes mitochondria after myocardial ischemia-reperfusion.

8. The application according to claim 7, characterized in that, The drug mentioned for preventing arrhythmias after myocardial ischemia-reperfusion is an injectable formulation.

9. The application according to claim 8, characterized in that, The drug for preventing and treating arrhythmias after myocardial ischemia-reperfusion also contains pharmaceutically acceptable excipients.

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