Construction method and application of arrhythmia zebrafish model with atrial enlargement and abnormal second intercalary structure

Abstract

The invention belongs to the technical field of animal model construction, and particularly relates to a construction method and application of an arrhythmia zebra fish model with atrial enlargement and abnormal second leap second structure, the model is a cyth3ae6 / e6 mutant zebra fish, sgRNA of a sixth exon of a target zebra fish cyth3a gene is designed, the cyth3a gene of a wild type zebra fish embryo is mutated by using a sgRNA and Cas9 protein mixture, and the target zebra fish cyth3a gene is obtained. Hybridizing the obtained F0-generation zebrafish with wild-type zebrafish, screening out F1-generation heterozygotes, performing internal crossing on the F1-generation heterozygotes, and screening out positive homozygote F2-generation zebrafish, namely a zebrafish model; the cyth3ae6 / e6 mutant zebrafish is constructed through cyth3a gene specific knockout for the first time, and the model construction method is simple, high in efficiency and high in success rate; the method can be used for evaluating or screening drugs for treating arrhythmia cardiomyopathy.

Description

Technical Field:

[0001] The present invention belongs to the technical field of animal model construction, and particularly relates to a method for constructing an arrhythmogenic zebrafish model with enlarged atria and abnormal intercalated disc structure, and an application thereof. This model can be used to screen drugs for treating arrhythmia. Background Art:

[0002] Arrhythmogenic cardiomyopathy (ACM) is a hereditary heart disease characterized by the gradual replacement of cardiomyocytes with fibroadipose tissue, starting from the epicardium and extending towards the endocardium. The occurrence of the disease will result in thinning of the ventricular wall accompanied by ventricular dilation, myocardial atrophy, aneurysms, syncope, and ventricular arrhythmia. These pathological changes can lead to sudden death in young people and athletes. In addition, physical exercise and competitive sports activities may trigger life-threatening ventricular arrhythmia, accelerating the progression of the disease and the risk of sudden cardiac death. The estimated prevalence of ACM in the general population is 1:2000 to 1:500. However, due to difficult or incorrect diagnosis, this frequency may be underestimated. This disease is distributed worldwide, but in Italy, especially in the Veneto region, the incidence rate is approximately 1:1000. ACM is a heterogeneous disease both clinically and genetically, mainly inherited in an autosomal dominant form with incomplete penetrance, but there are also recessive forms, such as Naxos syndrome and Carvajal syndrome. Currently, the management of ACM is mainly to delay the progression of the disease and prevent sudden cardiac death, and there is no cure for this life-threatening disease.

[0003] As an experimental animal model, zebrafish has attracted attention due to the similarity of its physiological and genetic characteristics and the convenience of experimental operations. Researchers use the CRISPR / Cas9 gene editing technology to knock out specific genes and simulate the function of the gene in the heart in order to study its pathogenesis and explore treatment methods. Currently, there is relatively little research on arrhythmogenic zebrafish models with enlarged atria and abnormal myocardial intercalated disc structure. Summary of the Invention:

[0004] The object of the present invention is to overcome the drawbacks of the prior art and provide a method for constructing an arrhythmogenic zebrafish model with enlarged atria and abnormal intercalated disc structure. Using zebrafish cyth3a as the target gene and the CRISPR / Cas9 technology, in vitro designed and synthesized cyth3a-specific sgRNA and Cas9 protein are microinjected into wild-type zebrafish fertilized eggs. After successive generations of screening, zebrafish mutants with specific knockout of the cyth3a gene are obtained. The arrhythmia phenotypes of the mutants are observed and passed on for conservation. The present invention can obtain genetically stable zebrafish mutants with arrhythmia. Utilizing the transparency and high fertility of zebrafish embryos, the mutants can be used to screen and verify the effects and impacts of different types of drugs for treating arrhythmia. The method for constructing this zebrafish animal model has important physiological significance and specificity for the screening of drugs for treating ACM diseases.

[0005] To achieve the above object, the present invention provides the following technical solution: A method for constructing an arrhythmogenic zebrafish model with enlarged atria and abnormal intercalated disc structure, designing an sgRNA targeting exon 6 of the zebrafish cyth3a gene, mutating the cyth3a gene of wild-type zebrafish embryos with a mixture of sgRNA and Cas9 protein, crossing the obtained F0 generation zebrafish with wild-type zebrafish, screening out F1 generation heterozygotes from the born zebrafish, inbreeding the F1 generation heterozygotes, and screening out positive homozygous F2 generation zebrafish from the born zebrafish, which is the arrhythmogenic zebrafish model with enlarged atria and abnormal intercalated disc structure; the specific steps are as follows:

[0006] (1) Synthesize an sgRNA targeting exon 6 of the zebrafish cyth3a gene, and the sequence of the sgRNA is as shown in SEQ ID NO:1, with the sequence 5’-TCAACTTGGTGCAAGCATTG-3’;

[0007] (2) Mix the sgRNA and Cas9 protein to prepare a Cas9 / sgRNA mixture;

[0008] (3) Introduce the Cas9 / sgRNA mixture into wild-type zebrafish embryos to obtain F0 generation zebrafish;

[0009] (4) Cross the F0 generation zebrafish with wild-type zebrafish, screen out F1 generation heterozygotes from the born zebrafish, inbreed the F1 generation heterozygotes, and screen out positive homozygous F2 generation zebrafish from the born zebrafish, which is the arrhythmogenic zebrafish model with enlarged atria and abnormal intercalated disc structure.

[0010] The present invention uses microinjection to introduce the Cas9 / sgRNA mixture into wild-type zebrafish embryos to obtain F0 generation zebrafish.

[0011] In the sgRNA / Cas9 mixture of the present invention, the concentration of sgRNA is 1.5 μM, and the concentration of Cas9 protein is 0.5 μg / μL.

[0012] In the present invention, the genotypes of F1 and F2 generation zebrafish were determined by extracting DNA from the caudal fin, using PCR amplification and Sanger sequencing.

[0013] The sequence of the sgRNA described in the present invention can also be methylated or otherwise modified.

[0014] The present invention also provides an arrhythmogenic zebrafish model with enlarged atria and abnormal intercalated disc structure, and this model is the cyth3a e6 / e6 mutant zebrafish, in which 13 bases are deleted from the cyth3a gene of zebrafish, resulting in premature termination of the expression of the cyth3a gene and abnormal expression of the Cyth3 protein; the arrhythmia characteristics are problems with the heart rhythm, abnormal P and T waves, and enlarged atria are found pathologically.

[0015] The said cyth3a e6 / e6 The mutant zebrafish was obtained by targeting gene knockout of 13 bases on exon 6 of the cyth3a gene by CRISPR / Cas9.

[0016] The present invention also provides the application of the arrhythmogenic zebrafish model with enlarged atria and abnormal intercalated disc structure in screening drugs for treating arrhythmogenic cardiomyopathy.

[0017] The method for constructing the cyth3a gene mutant zebrafish model provided by the present invention is to construct the cyth3a gene mutant zebrafish model through the CRISPR / Cas9 system; however, other conventional gene editing methods can also be used to obtain the cyth3a gene mutant zebrafish.

[0018] Compared with the prior art, the present invention for the first time constructs an arrhythmogenic zebrafish model with enlarged atria and abnormal myocardial intercalated disc structure by specifically knocking out the cyth3a gene. The model construction method is simple, with high efficiency and high success rate; it can be used for drug screening, providing great help and convenience for evaluating or screening drugs for treating arrhythmogenic cardiomyopathy.

[0019] Description of the drawings:

[0020] Figure 1 It is a schematic diagram of mutating the cyth3a gene of wild-type zebrafish fertilized eggs and an effective Sanger sequencing map of F0 generation mutant zebrafish involved in the present invention. Among them, A is the schematic diagram of gene mutation, and the dotted line indicates the deleted nucleotides; B is the Sanger sequencing map of F0 generation mutant zebrafish, and the red box indicates the appearance of overlapping peaks after the knockout site.

[0021] Figure 2 This invention relates to the gene sequencing results and protein mutation conditions of F2 generation zebrafish. Among them, A is a schematic diagram of the Sanger sequencing results of wild-type, heterozygous, and homozygous zebrafish in the F2 generation. The sequence within the box represents the region of the site in the wild-type sequence deleted in the mutant. The black vertical line indicates the position where peak doubling appears in the sequencing of heterozygotes and wild-types starting from this position; B is the Cyth3a protein and the truncated protein (secondary structure) after mutation; C is the spatial simulation structure of the Cyth3a protein. The red arrow points out the truncated site, and the red square indicates that this part of the structure is missing.

[0022] Figure 3 This is a schematic diagram of the heart function detection results of homozygous mutant zebrafish larvae involved in this invention. Among them, a is the white light phenotype of wild-type zebrafish larvae (WT) and cyth3a e6 / e6 When the homozygous mutant larvae develop to the 5th day, the atrium is swollen at the green asterisk, the heart structure is abnormally flipped. After dissection, it can be seen that the atrium (A) of the homozygous becomes larger and the ventricle (V) becomes smaller; b is the comparison of the heart rates (Bmp) of wild-type and homozygous mutants; c and d are the comparisons of the cross-sectional areas of the ventricles and atria of the hearts of wild-type and homozygous mutants respectively. e is the comparison of the fractional shortening (FS) of the hearts of wild-type and homozygous mutants.

[0023] Figure 4 This is a schematic diagram of the comparison results of the ultrasonic detection of heart functions of homozygous mutant zebrafish adults and wild-type adults involved in this invention. Among them, A is the heart function index measured in the B-ultrasound mode of adult fish; B is the echocardiogram in the systolic (left figure) and diastolic (right figure) phases of the heart; C is the heart pumping function index in the PW mode.

[0024] Figure 5 This is the representative electrocardiogram and statistical results of homozygous mutant zebrafish and wild-type adults involved in this invention. Among them, A is the representative ECG; B and C are the statistics of the heart rates, abnormal P waves, and arrhythmias in the ECG of the two groups of fish; D is the statistics of the time of each wave band in the electrocardiogram.

[0025] Figure 6 This is a schematic diagram of the maximum swimming speed results of wild-type and homozygous mutant zebrafish involved in this invention.

[0026] Figure 7 This invention relates to cyth3a at 3 months old e6 / e6Comparison results of heart sizes between mutants and wild-type control groups. Among them, A is a representative image of the excised heart, B is the quantification of the normalized ventricular surface area (VSA) versus body weight (BW / g), C is the quantification of the normalized atrial surface area (VSA) versus body weight (BW / g), D is a representative picture of the atrium measured in B-mode ultrasound, and E is the result of the quantification of the atrial surface area.

[0027] Figure 8 For 3-month-old cyth3a e6 / e6 HE and Masson staining sections of the hearts of mutant and wild-type control zebrafish. A is the result of the HE section, B is the quantification ratio of the cross-sectional areas of the atrium and ventricle, and C is the result of the Masson staining section.

[0028] Figure 9 For 3-month-old cyth3a e6 / e6 TEM images (2500×) and enlarged images (20000×) of the hearts of mutant zebrafish.

[0029] Figure 10 For cyth3a e6 / e6 Application results of mutant zebrafish. Among them, A is the heart rate statistics of 3-day-old embryos after using SB216763; B is the heart morphology of 3-day-old embryos after using SB216763; C is the protein expression of GSK3β and β-catenin in embryos after using SB216763. Specific implementation manner:

[0030] The technical solution of the present invention will be described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0031] Example 1:

[0032] This example relates to a method for constructing an arrhythmic zebrafish model with enlarged atria and abnormal myocardial intercalated disc structures. The specific steps are as follows:

[0033] (1) Synthesize sgRNA targeting exon 6 of the zebrafish cyth3a gene (NCBI: Gene ID 100002190). The sequence of the sgRNA is as shown in SEQ ID NO:1, and the sequence of SEQ ID NO:1 is 5’-TCAACTTGGTGCAAGCATTG-3’;

[0034] (2) Prepare an injection plate: Prepare a gel solution with an agarose mass concentration of 1.5% using embryo culture medium (0.292 g NaCl, 0.013 g KCl, 0.044 g CaCl2, 0.081 g MgSO4, pH 7.2) and agarose powder. Pour the agarose gel solution into a 10 cm cell culture plate while it is hot, and use an injection groove template to make a gel injection plate, and let it cool and solidify;

[0035] (3) Preparation of injection needles: Use a needle puller to heat and pull a capillary glass tube for injection to obtain a glass needle for injection. Cut the thin end of the glass needle according to the required amount during injection. Mix 5 μL of sgRNA with 5 μL of Cas9 protein. The final concentration of sgRNA is 1.5 μM, and the final concentration of Cas9 protein is 0.5 μg / μL. Incubate at 37 °C for 10 min. After obtaining the Cas9 / sgRNA mixture, add 0.2 μL of phenol red indicator and then inject it into the glass injection needle.

[0036] (4) Microinjection: Fix the injection needle on the injector, and adjust the droplet size through the adjustment knob on the injector. The maximum injection volume does not exceed 1 nl. Place the freshly fertilized wild-type zebrafish embryos collected into a culture dish containing embryo culture medium. Use a Pasteur pipette to suck the embryos into the injection dish, and gently arrange the fish eggs neatly with a yellow pipette tip. For formal injection, adjust the angle so that the liquid is injected into the cells at the 1-cell stage of the embryo or into the middle of the yolk. After all injections are completed, gently blow the embryos with a Pasteur pipette and then place them into a culture dish containing fresh embryo culture medium for incubation at a constant temperature of 28.5 °C to obtain F0 generation zebrafish. At the same time, retain some non-injected embryos of the same batch as the wild-type control group.

[0037] (5) PCR amplification: When the fertilized eggs develop to 3 days after injection, randomly pick 5 embryos from the injected group and non-injected control group respectively. Transfer the zebrafish embryos into a PCR tube, add 40 μL of NaOH with a concentration of 50 mM, and lyse at 95 °C in a PCR instrument for 30 min. After quickly mixing evenly with a vortex oscillator; then briefly oscillate and centrifuge at high speed for 10 s, and add 4 μL of Tris-HCl (pH = 8.0) to the lysate for neutralization. Vortex for 30 s, and the supernatant after centrifugation at high speed is the genomic DNA of the zebrafish embryos. Using it as a template, perform PCR amplification with the detection primers upstream and downstream of the target sequence (Primer F: 5’-TCGAGGCATCAATGAAGGAGGAG-3’, R: 5’-CGG TCTGGATTAAAGAAGGTGTGA-3’). The PCR reaction system and reaction conditions are shown in Table 1 and Table 2.

[0038] Table 1 PCR amplification reaction system

[0039]

[0040] Table 2 PCR reaction conditions

[0041]

[0042] (6) Sequencing to identify whether the sgRNA works: After detecting that there are no extra bands in the nucleic acid electrophoresis of the PCR product (the amplified target fragment is 186 bp), send it to a sequencing company for Sanger sequencing. If the sequencing result shows a single peak at the target site position, then no mutation has occurred; if the result shows chaotic peaks near the target site, then a mutation has occurred, the sgRNA is effective, and the F0 generation zebrafish is the mutant zebrafish needed. The detection results are as Figure 1 shown. It can be seen from Figure 1 B that compared with wild-type zebrafish, chaotic peaks appear near the target site in the F0 generation zebrafish, indicating that gene knockout is successful;

[0043] (7) Cross the F0 generation zebrafish with wild-type zebrafish to produce offspring F1. Raise them to adult fish. Cut the tails of 2-month-old F1 generation zebrafish to release DNA, and use PCR to amplify the target band to detect and verify whether the cyth3a gene has mutated. Select the stably inherited heterozygotes (missing 13 bp) in the F1 generation for subsequent experiments;

[0044] (8) According to the sequencing results, raise the F1 generation heterozygous zebrafish in the same tank. After they reach sexual maturity and mate, F2 generation zebrafish can be obtained; select wild-type, heterozygous (Cyth3a e6 / + ) and homozygous (Cyth3a e6 / e6 ) zebrafish through PCR and Sanger sequencing. The sequencing results are as Figure 2 shown. Compared with the wild-type, the homozygous has a 13-base deletion, resulting in premature termination of the expression of the cyth3a gene and abnormal expression of the CYTH3 protein ( Figure 2 ). Select the homozygous zebrafish with homozygous mutation of cyth3a in F2 as the arrhythmic zebrafish model with enlarged atria and abnormal myocardial intercalated disc structure for subsequent experiments and conservation.

[0045] Example 2:

[0046] This example involves an analysis experiment on the impact of cyth3a e6 / e6 mutant zebrafish on heart function. The specific experiment is as follows:

[0047] 1. Analysis of the heart function of cyth3a e6 / e6 mutant zebrafish larvae

[0048] (1) Obtaining of Cyth3a e6 / e6 mutant zebrafish larvae: Put one male and one female Cyth3a e6 / e6 homozygous adult fish in each mating tank, separate them with a baffle in the middle. Remove the baffle the next day to collect the fertilized eggs. Put the fertilized eggs into the embryo culture medium and incubate them at a constant temperature of 28.5 °C. Observe the heart morphology, measure the heart rate, measure the cross-sectional areas of the ventricle and atrium, and measure the heart ejection fraction when the larvae develop to the 5th day;

[0049] (2) Observation of the heart morphology of juvenile fish: Use a pipette to take out the juvenile fish and anesthetize it in 0.02% tricaine for 1 min. Place the fish on its side and fix it in 3% methylcellulose. Observe the heart morphology using a Zeiss Axioplan 2 differential interference camera lens with a magnification of 20 times. Under the microscope, it can be seen that the hearts of homozygous zebrafish embryos are malformed and the atria are enlarged. After dissection, it can be seen that the atria (A) of the homozygotes are enlarged and the ventricles (V) are smaller ( Figure 3 A);

[0050] (3) Heart rate measurement: Observe under the microscope where the heartbeat is clearly visible. Use a manual counter and timer to record the number of heartbeats of normal zebrafish juveniles and mutant juveniles within 30 s. Each embryo is repeated three times, and more than 5 embryos are repeated in each group. The results show that compared with the wild type, cyth3a e6 / e6 the heart rate of the mutant model is significantly reduced ( Figure 3 B);

[0051] (4) Measurement of the cross-sectional areas of the ventricles and atria of juvenile zebrafish: In the video of the juvenile fish heart, save the frames of ventricular and atrial diastole (VD, AD) as Tiff format, and then use Image J to open the photos of the maximum end-diastolic ventricle or the maximum atrium for analysis. 8 are measured in each group ( Figure 3 C and 3D), and the results show that cyth3a e6 / e6 the cross-sectional area of the ventricle of the mutant is significantly smaller than that of the wild type, and the cross-sectional area of the atrium is significantly larger than that of the wild type group;

[0052] (5) Measurement of the ejection fraction of the heart of juvenile zebrafish: Record a 20-s heart-beating video file. In the video file, use the arrow keys to move between frames. Save the frames of the minimum ventricular contraction (VS) and the maximum ventricular diastole (VD) as Tiff format. Measure the heart width at the maximum systolic and diastolic phases of the ventricle in pixels. 8 are measured in each group, and calculate the fractional shortening FS (%) of the ventricle = (width at diastole - width at systole) / (width at diastole) * 100 ( Figure 3 E), and the results show that compared with the wild type, cyth3a e6 / e6 the FS of the mutant is significantly reduced;

[0053] 2. Analysis of the heart function of adult cyth3a e6 / e6 mutant zebrafish

[0054] Perform heart function detection, electrocardiogram detection, swimming tunnel test, measurement of atrial and ventricular surface areas, and transmission electron microscopy detection on 3-month-old cyth3a e6 / e6 mutant zebrafish. The specific steps are as follows:

[0055] (1) Adult fish cardiac function detection: Use a Vevo 3100 high-frequency imaging system with 50 MHz (MX700) to measure and analyze the cardiac function indices of 3-month-old cyth3a e6 / e6 mutant zebrafish. Anesthetize zebrafish with 0.02% tricaine for 5 minutes, place them abdomen-up in a sponge, apply acoustic gel on the probe surface to provide sufficient coupling with the tissue interface, place the 50 MHz (MX700) sensor above the zebrafish to provide sagittal imaging of the heart. B-mode images are taken with an axial imaging field of view of 7.00 mm, a transverse imaging field of view of 4.73 mm, a frame rate of 281 Hz, and the transmission focus at the center of the heart. Use the data packet in the VevoLAB workstation for image quantification. Measure cardiac function phenotypes based on B-mode images using the following three indices: Ejection fraction (EF) = (EDV - ESV) / EDV; FS = (EDD - ESD) / EDD, FAC = (EDA - ESA) / EDA. EDV and ESV are the ventricular volumes at the end of diastole and systole of the ventricle, respectively. EDD and ESD are the vertical distances from the ventricular apex to the ventricular base line at the end of diastole and systole of the ventricle, respectively; EDA and ESA are defined as the ventricular areas at the end of diastole and systole of the ventricle, respectively. Measure ventricular dimensions from B-mode images using the following two indices: EDV / body weight (BW) and ESV / BW. Under the guidance of B-ultrasound mode, record PW Doppler signals to measure blood flow velocity, and record PW Doppler signals for about 4.84 s in the WT group and the cyth3a e6 / e6 mutant group and store them for offline analysis in MATLAB. To study cardiac hemodynamics, analyze the pulsed wave Doppler signals of passive (E-wave velocity) and active (A-wave velocity) ventricular filling during diastole. For each fish's indices, measure 3 - 5 independent cardiac cycles to obtain the average value. The results are as Figure 4 shown.

[0056] As can be seen from Figure 4 A and 4B, the ejection fraction (EF), fractional shortening (FS), fractional area change of the ventricle (FAC), ESV / BW, and EDV / BW of the hearts of 3-month-old wild-type zebrafish are approximately 54%, 19.5%, 31.9%, 1.2 μL / g, and 2.4 μL / g, respectively, while those of the cyth3a e6 / e6- mutant zebrafish are approximately 38%, 12.5%, 22%, 1.7 μL / g, and 2.5 μL / g, respectively, indicating that: compared with wild-type zebrafish, the cardiac pumping function of the cyth3a e6 / e6 mutant zebrafish is significantly weakened. PW analysis is shown in Figure 4 C, and it can be seen that the ejection time of the ventricle of the cyth3a e6 / e6- mutant zebrafish becomes longer and the systolic function decreases. Comprehensive analysis shows that cyth3ae6 / e6- The cardiac function of mutant zebrafish is significantly reduced compared to that of the wild type.

[0057] (2) Electrocardiogram (ECG) detection of adult fish: One week before collecting the electrocardiogram, microsurgery was performed on the fish under a stereomicroscope to remove the silver epithelial layer under the skin outside the heart. One week after microsurgery recovery, the fish was anesthetized with 0.02% tricaine for 5 minutes, transferred to a dented sponge, placed in the prone position, and a few drops of fish-raising water were added to the surface skin of the fish to keep the surface of the fish moist. The iWorx system (IWX214, iWorx Systems, Inc) was used to record the electrocardiogram for 4 minutes, and then the fish was transferred to the system fish-raising water for recovery. The results are as Figure 5 shown in A-5C.

[0058] From the representative electrocardiogram Figure 5 A and the statistical results 5B-5C, it can be seen that cyth3a e6 / e6 mutant zebrafish will have arrhythmia, abnormal P waves, and elevated T wave lengths; indicating that the constructed cyth3a e6 / e6 mutant zebrafish has no change in the average heart rate but has arrhythmia, ventricular hypertrophy, or myocardial infarction. From Figure 5 D, it can be analyzed that compared with the wild type, the P-R and R-R wave bands of cyth3a e6 / e6 mutant zebrafish increase in time, but not significantly, while the time of QRS is significantly reduced, and the duration of QTc and T waves is significantly increased.

[0059] (3) Swimming tunnel test: cyth3a e6 / e6 mutant zebrafish were raised together with age-matched wild-type zebrafish. Before measuring the swimming ability, all fish were fasted for 24 h; to evaluate the swimming ability of adult fish, 3-month-old zebrafish were placed in a swimming tunnel with an initial water speed of 9 cm / s for a 20-min acclimation period, and then the water flow was increased in stages at a speed of 8.66 cm / s (Ui) every 150 s (Tii) until all fish were exhausted. The maximum swimming speed (Uii) and the duration of the maximum speed (Ti) of each fish were recorded. The formula for calculating the critical swimming ability (Ucrit) is: Ucrit = Uii + [Ui × (Ti / Tii)], and then Ucrit was normalized to the body length (BL) of the corresponding individual. The results are as Figure 6 shown.

[0060] From Figure 6 it can be seen that the critical swimming ability of cyth3a e6 / e6- mutant zebrafish is significantly decreased.

[0061] (4) Measuring the atrial and ventricular surface areas: After anesthetizing 3-month-old zebrafish and weighing them (BW), the heart of a single zebrafish was dissected and imaged with a millimeter ruler under a Leica stereomicroscope to measure the ventricular surface area (VSA) and atrial surface area (ASA). Additionally, to further confirm the atrial area in the in vivo state, it was measured using a 50 MHz (MX700) Vevo3100 high-frequency imaging system in B-mode ultrasound. The results are as Figure 7 shown.

[0062] As can be seen from Figure 7 it, the ventricular surface area of the cyth3a e6 / e6 mutant zebrafish heart was significantly smaller than that of the wild type after excision ( Figure 7 A and 7B), and the atrial surface area was significantly larger than that of the wild type ( Figure 7 A and 7C). To further verify the size of the mutant atrium, combined with the results in the B-mode ultrasound state, it can be seen that in the in vivo state, the atrium of the mutant zebrafish was also significantly enlarged ( Figure 7 D and 7E). This indicates that the cyth3a e6 / e6 mutant zebrafish exhibited an atrial dilation phenotype.

[0063] (5) Histopathology: After euthanizing 3-month-old zebrafish, the hearts were immediately fixed with 4% formaldehyde fixative, paraffin-embedded, and the sections were stained with hematoxylin and eosin (H&E) and masson. Images of the apex region were taken using a Ni-U imaging system (Nikon). The results are as Figure 8 shown.

[0064] Figure 8 A and Figure 8 B show that the ratio of the atrial / ventricular cross-sectional area of the cyth3a e6 / e6 mutant zebrafish was significantly higher than that of the wild type, and Figure 8 the masson staining in C did not show myocardial fibrosis after the cyth3a gene mutation. This further indicates that knocking out the cyth3a gene leads to an enlarged atrium.

[0065] (6) Transmission electron microscopy (TEM): In the TEM study, the zebrafish hearts taken were immediately fixed in a fixative (4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2) at room temperature for 1 h and then placed at 4°C overnight. The fixed samples were subsequently processed by Wuhan Faber Biotechnology Co., Ltd. and imaged using an HT7800 transmission electron microscope. The results are as Figure 9 shown.

[0066] As can be confirmed from Figure 9 it, for 3-month-old cyth3a e6 / e6In the myocardium of mutant zebrafish, mitochondria showed swelling and degeneration (green triangle), and some showed vacuolization (red triangle). The widened intercalated disc space could also be clearly seen. The results of electron microscopy revealed cyth3a e6 / e6 In mutant zebrafish, lesions occurred at the cell junctions.

[0067] Example 3:

[0068] This example relates to the application of cyth3a e6 / e6 mutant zebrafish, specifically:

[0069] Using the cyth3a constructed in Example 1 e6 / e6 mutant zebrafish to screen for drugs for treating ACM disease, and drugs that can improve or rescue the phenotypes of cyth3a e6 / e6 mutant zebrafish larvae such as arrhythmia and morphological abnormalities are the target drugs.

[0070] Set up a wild-type control group (WT), a cyth3a e6 / e6 mutant control group (cyth3a e6 / e6 ), a wild-type drug treatment group (WT+SB216763) and a cyth3a e6 / e6 mutant drug treatment group (cyth3a e6 / e6 +SB216763). Use a 6-well cell culture plate, place 20 zebrafish embryos in each well, and culture at 28.5°C. Prepare a 10 mM concentration of SB216763 solution with zebrafish culture water. At 1 day old of zebrafish embryos, the wild-type drug treatment group and the cyth3a e6 / e6 mutant drug treatment group were administered at a final concentration of 4.5 μM. Observe the changes in phenotypes such as the morphology, heart rate, and heart morphology of cyth3a e6 / e6 mutant zebrafish according to the method in Example 2 and detect the effect of the SB216763 drug by Western Blot.

[0071] The results showed that under the treatment of a 4.5 μM concentration of SB216763 solution, the heart rate of cyth3a e6 / e6 mutant zebrafish was significantly higher than that of the wild-type drug treatment group, but still relatively slow compared with the wild-type control group ( Figure 10 A). Autopsy found that the atrium of cyth3a e6 / e6 mutant zebrafish became significantly smaller after using the SB216763 drug ( Figure 10 B). The Western Blot results showed that SB216763 could reduce the phosphorylation of GSK3β and β-catenin ( Figure 10C). The above results indicate that SB216763 has a certain alleviating effect on the symptoms of the zebrafish model of the present invention and inhibits the phosphorylation of GSK3β and β-catenin. Therefore, inhibiting β-catenin can be used as a potential drug target for treating ACM disease.

Claims

1. An arrhythmogenic zebrafish model with enlarged atria and abnormal intercalated disc structure, characterized in that, This model is cyth3a e6 / e6 Mutant zebrafish.

2. The arrhythmogenic zebrafish model with enlarged atria and abnormal intercalated disc structure according to claim 1, characterized in that, The zebrafish model is a mutant zebrafish in which 13 bases are deleted from the zebrafish cyth3a gene, resulting in premature termination of the expression of the cyth3a gene and abnormal expression of the CYTH3 protein.

3. A method for constructing an arrhythmogenic zebrafish model with enlarged atrium and abnormal intercalated disc structure, characterized in that, Design an sgRNA targeting exon 6 of the zebrafish cyth3a gene, mutate the cyth3a gene of wild-type zebrafish embryos with a mixture of sgRNA and Cas9 protein, hybridize the obtained F0 generation zebrafish with wild-type zebrafish, and screen for F1 generation heterozygotes from the newly born zebrafish; perform inbreeding on the F1 generation heterozygotes, and screen for positive homozygous F2 generation zebrafish from the newly born zebrafish, which are the arrhythmic zebrafish models with enlarged atria and abnormal intercalated disc structure.

4. The method for constructing an arrhythmogenic zebrafish model with enlarged atrium and abnormal intercalated disc structure according to claim 3, wherein Introduce the mixture of the sgRNA and Cas9 protein into wild-type zebrafish embryos by microinjection to obtain F0 generation zebrafish.

5. The method for constructing an arrhythmogenic zebrafish model with enlarged atrium and abnormal intercalated disc structure according to claim 3, wherein, In the sgRNA / Cas9 protein mixture, the concentration of sgRNA is 1.5 μM, and the concentration of Cas9 protein is 0.5 μg / μL.

6. Use of the arrhythmic zebrafish model according to claims 1-2 or the arrhythmic zebrafish model with enlarged atria and abnormal intercalated disc structure constructed by the construction method according to any one of claims 3-5 in screening drugs for treating arrhythmogenic cardiomyopathy.

Images