A method for constructing and applying a zebrafish model of arrhythmia with atrial enlargement and intercalated disc structural abnormalities.

Abstract

This invention belongs to the field of animal model construction technology, specifically relating to a method and application for constructing a zebrafish model of arrhythmia with atrial enlargement and abnormal intercalated disc structure. This model is cyth3a. e6 / e6 A mutant zebrafish model was developed by designing an sgRNA targeting exon 6 of the zebrafish cyth3a gene. The cyth3a gene in wild-type zebrafish embryos was mutated using a mixture of sgRNA and Cas9 protein. The resulting F0 generation zebrafish were crossed with wild-type zebrafish, and heterozygotes in the F1 generation were selected. These heterozygotes were then inbred, and positive homozygotes in the F2 generation were selected, thus forming the zebrafish model. This is the first time that a cyth3a gene-specific knockout model has been constructed. e6 / e6 The mutant zebrafish model is simple, efficient, and has a high success rate; it can be used to evaluate or screen drugs for the treatment of arrhythmic cardiomyopathy.

Description

Technical Field:

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method and application of constructing a zebrafish model of arrhythmia with atrial enlargement and abnormal intercalated disc structure. This model can be used to screen drugs for the treatment of arrhythmias. Background technology:

[0002] Arrhythmic cardiomyopathy (ACM) is a hereditary heart disease characterized by the gradual replacement of cardiomyocytes with fibroadipose tissue, starting in the epicardium and extending towards the endocardium. The disease manifests as thinning of the ventricular walls accompanied by ventricular dilation, myocardial atrophy, aneurysms, syncope, and ventricular arrhythmias. These pathological changes can lead to sudden death in young people and athletes. Furthermore, physical exercise and competitive sports activities can trigger life-threatening ventricular arrhythmias, accelerating disease progression and increasing the risk of sudden cardiac death. The estimated prevalence of ACM in the general population is 1 in 2000 to 1 in 500; however, this frequency may be underestimated due to diagnostic difficulties or errors. The disease is distributed worldwide, but in Italy, particularly in the Veneto region, the incidence is approximately 1 in 1000. ACM is clinically and genetically heterogeneous, primarily inherited in an incompletely penetrating autosomal dominant form, but recessive forms also exist, such as Naxos syndrome and Carvajal syndrome. Currently, the management of ACM mainly focuses on slowing disease progression and preventing sudden cardiac death, as there is no cure for this life-threatening disease.

[0003] Zebrafish have attracted attention as a laboratory animal model due to their similarity in physiological and genetic characteristics and the ease of experimental manipulation. Researchers use CRISPR / Cas9 gene editing technology to knock out specific genes and mimic their function in the heart in order to study their pathogenesis and explore treatment methods. Currently, there is limited research on zebrafish models of arrhythmias characterized by atrial enlargement and abnormal intercalated disc structure. Summary of the Invention:

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for constructing a zebrafish model of arrhythmia with atrial enlargement and abnormal intercalated disc structure. Using zebrafish cyth3a as the target gene, and employing CRISPR / Cas9 technology, a designed and synthesized cyth3a-specific sgRNA and Cas9 protein are microinjected into wild-type zebrafish fertilized eggs. Through successive generations of screening, cyth3a gene-specific knockout zebrafish mutants are obtained. The arrhythmia phenotype of the mutants is observed and the mutants are passaged and preserved. This invention can obtain genetically stable arrhythmic zebrafish mutants. Utilizing the transparency and high reproductive capacity of zebrafish embryos, these mutants can be used to screen and verify the effects and influences of different types of drugs for treating arrhythmia. This method for constructing a zebrafish animal model has significant physiological importance and specificity for screening drugs for the treatment of arrhythmia.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for constructing a zebrafish model of arrhythmia with atrial enlargement and intercalated disc structure abnormalities. The method involves designing an sgRNA targeting exon 6 of the zebrafish cyth3a gene, mutating the cyth3a gene in wild-type zebrafish embryos using a mixture of sgRNA and Cas9 protein, crossing the obtained F0 generation zebrafish with wild-type zebrafish, selecting F1 generation heterozygotes from the offspring, performing inbreeding on the F1 generation heterozygotes, and selecting positive homozygotes for the F2 generation zebrafish, which constitute the zebrafish model of arrhythmia with atrial enlargement and intercalated disc structure abnormalities. The specific steps are as follows:

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

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

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

[0009] (4) Cross F0 generation zebrafish with wild-type zebrafish, select F1 generation heterozygotes from the born zebrafish, perform in-breeding of F1 generation heterozygotes, select positive homozygotes F2 generation zebrafish from the born zebrafish, which is the arrhythmia zebrafish model with enlarged atria and abnormal intercalated disc structure.

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

[0011] The sgRNA / Cas9 mixture of the present invention has a concentration of 1.5 μM for sgRNA and a concentration of 0.5 μg / uL for Cas9 protein.

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

[0013] The sgRNA sequence described in this invention can also be modified by methylation or other modifications.

[0014] This invention also provides a zebrafish model of arrhythmia with atrial enlargement and intercalated disc structural abnormalities, which is cyth3a. e6 / e6 The mutant zebrafish is a zebrafish with a 13-base deletion in the cyth3a gene, resulting in premature termination of cyth3a gene expression and abnormal Cyth3 protein expression; the arrhythmia is characterized by problems with heart rhythm, abnormal P waves and T waves, and pathological findings of atrial enlargement.

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

[0016] The present invention also provides the application of the arrhythmic zebrafish model with atrial enlargement and intercalated disc structural abnormalities in screening drugs for the treatment of arrhythmic cardiomyopathy.

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

[0018] Compared with existing technologies, this invention is the first to construct an arrhythmic zebrafish model with atrial enlargement and abnormal intercalated disc structure of the myocardium through specific knockout of the cyth3a gene. The model construction method is simple, efficient and has a high success rate; it can be used for drug screening, which provides great help and convenience for drug research to evaluate or screen drugs for the treatment of arrhythmic cardiomyopathy.

[0019] Instruction manual illustrations:

[0020] Figure 1 This invention relates to a schematic diagram of mutation of the cyth3a gene in wild-type zebrafish fertilized eggs and an effective Sanger sequencing diagram of F0 generation mutant zebrafish. In the diagram, A is a schematic diagram of gene mutation, with dashed lines indicating deleted nucleotides; B is a Sanger sequencing diagram of F0 generation mutant zebrafish, with red boxes indicating overlapping peaks after knockout.

[0021] Figure 2 This invention relates to the gene sequencing results and protein mutation status of F2 generation zebrafish. A is a schematic diagram of the Sanger sequencing results of F2 generation wild-type, heterozygous, and homozygous zebrafish. The sequence in the box represents the region of the deleted wild-type sequence in the mutant. The black vertical line indicates the position where overlapping peaks appear in the sequencing of heterozygous and wild-type. 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 to the truncated site, and the red box indicates that this structure is missing.

[0022] Figure 3 This is a schematic diagram of the cardiac function test results of homozygous mutant zebrafish juveniles involved in the present invention, where a represents wild-type zebrafish juveniles (WT) and cyth3a. e6 / e6 On day 5, the homozygous mutant juveniles exhibited a white phenotype, with enlarged atria and malformed, inverted heart structures indicated by green asterisks. Dissection revealed enlarged atria (A) and smaller ventricles (V) in the homozygous mutants. b shows a comparison of heart rate (Bmp) between wild-type and homozygous mutants; c and d show a comparison of the cross-sectional areas of the ventricles and atria in wild-type and homozygous mutants, respectively. e shows a comparison of the fractional shortening (FS) of the heart between wild-type and homozygous mutants.

[0023] Figure 4 This diagram illustrates the comparison of cardiac function between homozygous mutant adult zebrafish and wild-type adult zebrafish according to the present invention. In the diagram, A represents cardiac function indicators of adult fish measured in B-mode ultrasound; B represents echocardiograms during cardiac systole (left) and diastole (right); and C represents cardiac pumping function indicators in PW mode.

[0024] Figure 5 The present invention relates to representative electrocardiograms and statistical results of homozygous mutant zebrafish and wild-type adult fish, wherein A is a representative ECG; B and C show the abnormalities in heart rate, P wave and arrhythmia in the two groups of fish in the ECG; and D shows the time of each wave segment in the statistical ECG.

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

[0026] Figure 7 This invention relates to 3-month-old cyth3a e6 / e6Comparison of heart size between mutant and wild-type control groups, where A is a representative image of isolated heart, B is the normalized ventricular surface area (VSA) and the quantification of body weight (BW / g), C is the normalized atrial surface area (VSA) and the quantification of body weight (BW / g), D is a representative image of the atrium measured in B-mode ultrasound, and E is the result of quantification of atrial surface area.

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

[0028] Figure 9 For 3-month-old cyth3a e6 / e6 TEM image (2500×) and magnified view (20000×) of the heart of mutant zebrafish.

[0029] Figure 10 for cyth3a e6 / e6 Results of the application of mutant zebrafish: A shows the heart rate statistics of embryos 3 days after using SB216763; B shows the heart morphology of embryos 3 days after using SB216763; and C shows the protein expression of GSK3β and β-catenin in embryos after using SB216763. Detailed implementation method:

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

[0031] Example 1:

[0032] This embodiment relates to a method for constructing a zebrafish model of arrhythmia with atrial enlargement and abnormal intercalated disc structure in the myocardium. The specific steps are as follows:

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

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

[0035] (3) Preparation of injection needle: Heat and pull open the capillary glass tube for injection using a needle puller to obtain a glass needle for injection. Cut the thin end of the glass needle according to the required amount before injection. Mix 5 μL of sgRNA and 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 / uL. Incubate at 37℃ for 10 min to obtain the Cas9 / sgRNA mixture. Add 0.2 μL of phenol red indicator and inject into the glass injection needle.

[0036] (4) Microinjection: Fix the injection needle on the syringe and adjust the droplet size by adjusting the knob on the syringe. The maximum injection volume should not exceed 1 ngl. Place the collected wild-type zebrafish embryos that have just been fertilized into a culture dish containing embryo culture medium. Use a Pasteur tube to aspirate the embryos and pour them into the injection dish. Use a yellow pipette tip to gently arrange the fish eggs neatly. Perform the injection and adjust the angle so that the liquid is injected into the cells of the 1-cell stage of the embryo or into the middle of the yolk. After all the injections are completed, use a Pasteur tube to gently blow up the embryos and place them into a culture dish containing fresh embryo culture medium. Incubate at a constant temperature of 28.5℃ to obtain F0 generation zebrafish. At the same time, retain some uninjected embryos from the same batch as a wild-type control group.

[0037] (5) PCR amplification: After injection, when the fertilized eggs developed to 3 days, 5 embryos from the injection group and 5 embryos from the non-injection control group were randomly aspirated. The zebrafish embryos were transferred to PCR tubes, and 40 μL of 50 mM NaOH was added. The mixture was lysed at 95 °C for 30 min. After vortexing, the mixture was quickly mixed. Then, the mixture was briefly centrifuged for 10 s. 4 μL of Tris-HCl (pH = 8.0) was added to the lysate for neutralization. The mixture was vortexed for 30 s. The supernatant after centrifugation was the genomic DNA of the zebrafish embryos. Using this DNA as a template, PCR amplification was performed using upstream and downstream detection primers for 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 determine if sgRNA is functional: After the PCR product is tested by nucleic acid electrophoresis and no impurities are detected (the amplified target fragment is 186 bp), it is sent to a sequencing company for Sanger sequencing. If the sequencing result shows a single peak at the target site, no mutation has occurred; if the result shows random peaks near the target site, a mutation has occurred, the sgRNA is effective, and the F0 generation zebrafish is the desired mutant zebrafish. The test results are as follows: Figure 1 As shown. From Figure 1 B shows that the F0 generation zebrafish exhibited disordered peaks near the target site compared to wild-type zebrafish, indicating that the gene knockout was successful.

[0043] (7) F0 generation zebrafish were crossed with wild-type zebrafish to produce offspring F1. The offspring F1 were raised to adulthood. The DNA of F1 generation zebrafish was released after tail cutting at 2 months of age. The target band was amplified by PCR to verify whether the cyth3a gene was mutated. Stable heterozygotes (13bp deletion) in F1 generation were selected for subsequent experiments.

[0044] (8) Based on the sequencing results, F1 generation heterozygous zebrafish were kept in the same tank and mated after reaching sexual maturity to obtain F2 generation zebrafish; wild-type and heterozygous (Cyth3a) zebrafish were selected by PCR and Sanger sequencing. e6 / + ) and homozygotes (Cyth3a) e6 / e6 The sequencing results of zebrafish are as follows: Figure 2 As shown, compared with the wild type, the homozygote has a 13-base deletion, which leads to premature termination of cyth3a gene expression and abnormal CYTH3 protein expression. Figure 2 Homozygous zebrafish with homozygous cyth3a mutations in F2 were selected as a model of arrhythmia with enlarged atria and abnormal intercalated myocardial disc structure for subsequent experiments and conservation.

[0045] Example 2:

[0046] This embodiment involves cyth3a e6 / e6 An analytical experiment was conducted to analyze the effects of mutant zebrafish on cardiac function. The specific experiment was as follows:

[0047] 1. cyth3a e6 / e6 Cardiac function analysis of mutant zebrafish juveniles

[0048] (1)Cyth3a e6 / e6 Acquisition of mutant zebrafish fry: Place one male and one female cyth3a in each mating tank. e6 / e6 Homozygous adult fish were separated by a baffle. The baffle was removed on the second day to collect the fertilized eggs. The fertilized eggs were placed in embryo culture medium and cultured at a constant temperature of 28.5℃. On the 5th day of the development of the juvenile fish, the heart morphology was observed, the heart rhythm was measured, the cross-sectional area of ​​the ventricle and atrium was measured, and the ejection fraction of the heart was measured.

[0049] (2) Observation of the heart morphology of juvenile fish: Juvenile fish were anesthetized for 1 minute in 0.02% tricaine using a pipette. They were then fixed in 3% methylcellulose with their sides facing upwards. The heart morphology was observed using a Zeiss Axioplan 2 differential interferometer camera lens with 20x magnification. Under the microscope, heart malformations and enlarged atria were observed in homozygous zebrafish embryos. After dissection, enlarged atria (A) and smaller ventricles (V) were observed in homozygous embryos. Figure 3 A);

[0050] (3) Heart rate measurement: Under a microscope, the heartbeats were clearly visible. A manual counter and timer were used to record the number of heartbeats in normal zebrafish juveniles and mutant juveniles over 30 seconds. Each embryo was repeated three times, and each group had at least five embryos repeated. The results showed that, compared with the wild type, cyth3a… e6 / e6 The mutant model showed a significant decrease in heart rate. Figure 3 B);

[0051] (4) Measurement of ventricular and atrial cross-sectional areas in juvenile zebrafish: From the heart videos of juvenile zebrafish, the diastolic (VD, AD) frames of the ventricles and atria were saved as TIFF format, and then the images of the ventricles at maximum end-diastole or the atria at maximum size were opened with ImageJ for analysis. Eight frames were measured in each group. Figure 3 (C and 3D), the results show cyth3a e6 / e6 The ventricular cross-sectional area of ​​the mutant was significantly smaller than that of the wild type, while the atrial cross-sectional area was significantly larger than that of the wild type.

[0052] (5) Measurement of cardiac ejection fraction in juvenile zebrafish: A 20-second video file of heartbeats was recorded. Arrow keys were used to navigate between frames in the video file. Minimum ventricular contraction (VS) and maximum ventricular diastole (VD) frames were saved as TIFF files. The ventricular width during maximum systole and diastole was measured in pixels, with 8 frames per group. The ventricular shortening fraction FS(%) was calculated as: (diastolic width - systolic width) / (diastolic width) * 100( Figure 3 E), the results showed that, compared with the wild type, cyth3a e6 / e6 The mutant showed a significantly reduced FS;

[0053] 2. cyth3a e6 / e6 Cardiac function analysis of adult mutant zebrafish

[0054] For 3 months of cyth3a e6 / e6 The mutant zebrafish underwent cardiac function testing, electrocardiogram testing, swimming tunneling test, measurement of atrial and ventricular surface areas, and transmission electron microscopy examination. The specific steps are as follows:

[0055] (1) Cardiac function test of adult fish: The cardiac function of cyth3a fish over a period of 3 months was measured and analyzed using a 50MHz (MX700) Vevo 3100 high-frequency imaging system. e6 / e6 Cardiac function parameters of mutant zebrafish. Zebrafish were anesthetized for 5 minutes with 0.02% tricaine, placed ventrally in a sponge, and an acoustic gel was applied to the probe surface to provide adequate coupling with the tissue interface. A 50MHz (MX700) sensor was placed above the zebrafish to provide sagittal imaging of the heart. B-mode images were obtained with an axial imaging field of view of 7.00 mm and a lateral imaging field of view of 4.73 mm, a frame rate of 281 Hz, and a transmission focus at the center of the heart. Image quantification was performed using data packets in the VevoLAB workstation. Cardiac function phenotype was measured based on the B-mode images using the following three parameters: ejection fraction (EF) = (EDV - ESV) / EDV; FS = (EDD - ESD) / EDD; FAC = (EDA - ESD) / EDA. EDV and ESV are the ventricular volumes at end-diastole and end-systole, respectively. EDD and ESD are the vertical distances from the ventricular apex to the ventricular basement line at end-diastole and end-systole, respectively; EDA and ESA are defined as the ventricular area at end-diastole and end-systole, respectively. Ventricular size was measured from B-mode images using two metrics: EDV / body weight (BW) and ESV / BW. PW Doppler signals were recorded under ultrasound guidance to measure blood flow velocity in the WT group and cyth3a. e6 / e6 PW Doppler signals of approximately 4.84 s were recorded in the mutant group and stored for offline analysis using MATLAB. To investigate cardiac hemodynamics, pulse-wave Doppler signals of passive (E-wave velocity) and active (A-wave velocity) ventricular filling during diastole were analyzed. For each fish, measurements were taken over 3–5 independent cardiac cycles to obtain average values. Results are as follows: Figure 4 As shown.

[0056] from Figure 4 As shown in A and 4B, the cardiac ejection fraction (EF), fractional shortening (FS), fractional change in ventricular area (FAC), ESV / BW, and EDV / BW of adult wild-type zebrafish at 3 months of age were approximately 54%, 19.5%, 31.9%, 1.2 μL / g, and 2.4 μL / g, respectively, while cyth3a e6 / e6- The EF, FS, SV, and FAC of the mutant zebrafish were approximately 38%, 12.5%, 22%, 1.7 μL / g, and 2.5 μL / g, respectively, indicating that compared with wild-type zebrafish, cyth3a... e6 / e6 The mutant zebrafish exhibited significantly reduced cardiac pumping function. PW analysis showed... Figure 4 C, see cyth3a e6 / e6- Mutant zebrafish exhibit prolonged ventricular ejection time and reduced contractile function. Comprehensive analysis suggests that cyth3a...e6 / e6- The mutant zebrafish had significantly reduced heart function compared to the wild type.

[0057] (2) Electrocardiogram (ECG) testing in adult fish: One week prior to ECG collection, the fish underwent microsurgery under a dissecting microscope to remove the silvery epithelial layer beneath the skin outside the heart. One week after microsurgery recovery, the fish were anesthetized with 0.02% tricaine for 5 minutes, transferred to a dented sponge, placed in a prone position, and a few drops of fish tank water were applied to the surface of the fish to maintain surface moisture. ECG recordings were performed for 4 minutes using an iWorx system (IWX214, iWorx Systems, Inc.). The fish were then transferred to the system's fish tank water for further recovery. Results are as follows: Figure 5 As shown in A-5C.

[0058] From representative electrocardiograms Figure 5 As can be seen from A and statistical results 5B-5C, cyth3a e6 / e6 Mutant zebrafish exhibit arrhythmia, abnormal P waves, and elevated T wavelengths; this indicates that the constructed cyth3a... e6 / e6 The mutant zebrafish maintained the same average heart rate, but exhibited arrhythmias, ventricular hypertrophy, or myocardial infarction. From Figure 5 Analysis of D reveals that, compared to the wild type, cyth3a e6 / e6 The mutant zebrafish showed increased, but not significant, PR and RR durations, but significantly decreased QRS duration, while QTc and T durations were significantly increased.

[0059] (3) Swimming tunnel test: cyth3a e6 / e6 Mutant zebrafish were reared together with age-matched wild-type zebrafish. All fish were fasted for 24 hours before swimming ability testing. To evaluate the swimming ability of adult fish, 3-month-old zebrafish were placed in a swimming tunnel with an initial water velocity of 9 cm / s for a 20-minute acclimatization period. The water flow was then gradually increased in stages at a rate of 8.66 cm / s (Ui) every 150 s (Tii) until all fish were exhausted. The maximum swimming speed (Uii) and the duration of maximum speed (Ti) for each fish were recorded. The critical swimming ability (Ucrit) was calculated using the formula: Ucrit = Uii + [Ui × (Ti / Tii)], and then Ucrit was normalized to the corresponding individual's body length (BL). The results are as follows: Figure 6 As shown.

[0060] from Figure 6 It can be seen that cyth3a e6 / e6- The critical swimming ability of mutant zebrafish is significantly reduced.

[0061] (4) Measurement of atrial and ventricular surface areas: After anesthetizing zebrafish for 3 months, they were weighed (BW), and then the hearts of individual zebrafish were dissected. The ventricular surface area (VSA) and atrial surface area (ASA) were measured using millimeter-scale imaging on a Leica stereomicroscope. To further confirm the atrial area in vivo, measurements were taken using a 50MHz (MX700) Vevo3100 high-frequency imaging system in ultrasound mode. The results are as follows: Figure 7 As shown.

[0062] from Figure 7 It can be seen that cyth3a e6 / e6 The ventricular surface area of ​​the mutant zebrafish heart was significantly smaller than that of the wild type after being removed from the body. Figure 7 A and 7B), 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 under ultrasound conditions, it can be seen that the atria of the mutant zebrafish are also significantly enlarged in vivo. Figure 7 (D and 7E). Explanation of cyth3a e6 / e6 Mutant zebrafish exhibited an atrial dilatation phenotype.

[0063] (5) Histopathology: Hearts of zebrafish euthanized at 3 months of age were immediately fixed with 4% formaldehyde fixative, embedded in paraffin, and sections were stained with hematoxylin and eosin (H&E) and masson stained. Images of the vertex region were captured using a Ni-U imaging system (Nikon). Results are as follows: Figure 8 As shown.

[0064] Figure 8 A and Figure 8 B shows that cyth3a e6 / e6 The ratio of atrium to ventricle cross-sectional area in mutant zebrafish was significantly higher than that in wild-type zebrafish. Figure 8 Masson staining of C cells did not show myocardial fibrosis following the cyth3a gene mutation. This further suggests that cyth3a gene knockout leads to atrial enlargement.

[0065] (6) Transmission Electron Microscopy (TEM): In the TEM study, zebrafish hearts were immediately fixed at room temperature in a fixative solution (0.1M pH 7.2 phosphate buffer containing 4% paraformaldehyde and 1% glutaraldehyde) for 1 hour, and then incubated overnight at 4°C. The fixed samples were subsequently processed by Wuhan Fabre Biotechnology Co., Ltd., and imaged using an HT7800 transmission electron microscope. Results are as follows: Figure 9 As shown.

[0066] from Figure 9 It has been confirmed that cyth3a is 3 months old. e6 / e6In mutant zebrafish myocardium, mitochondria exhibited swelling and degeneration (green triangles), with some showing vacuolation (red triangles), and widening of the intercalated disc space was clearly visible. Electron microscopy results revealed cyth3a e6 / e6 The mutant zebrafish exhibits lesions at the cell junctions.

[0067] Example 3:

[0068] This embodiment involves cyth3a e6 / e6 The applications of mutant zebrafish are as follows:

[0069] cyth3a constructed using Example 1 e6 / e6 Screening mutant zebrafish for drugs to treat ACM disease could improve or save cyth3a. e6 / e6 Drugs that address the phenotypes of mutant zebrafish juveniles, such as arrhythmia and morphological abnormalities, are the target drugs.

[0070] A wild-type control group (WT) and cyth3a were set up. e6 / e6 mutant control group (cyth3a) e6 / e6 ), wild-type drug treatment group (WT+SB216763) and cyth3a e6 / e6 mutant drug treatment group (cyth3a) e6 / e6 Zebrafish embryos (+SB216763) were cultured in 6-well plates at 28.5°C with 20 embryos placed in each well. A 10 mM solution of SB216763 was prepared using zebrafish culture water. At 1 day of age, wild-type embryos and cyth3a were cultured. e6 / e6 The mutant drug treatment group was administered the drug at a final concentration of 4.5 μM, and cyth3a was observed according to the method described in Example 2. e6 / e6 The effects of SB216763 drug were detected by Western Blot analysis of phenotypic changes in mutant zebrafish, including morphology, heartbeat, and heart morphology.

[0071] The results showed that, under treatment with 4.5 μM SB216763 solution, cyth3a e6 / e6 The heart rate of the mutant zebrafish was significantly higher than that of the wild-type drug-treated group, but it was still relatively slow compared to the wild-type control group. Figure 10 A) The autopsy revealed cyth3a e6 / e6 Mutant zebrafish showed a significant reduction in atrial size after treatment with SB216763. Figure 10 B), Western blotting 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 serve as a potential drug target for the treatment of ACM disease.

Claims

1. A method for constructing a zebrafish model of arrhythmia with atrial enlargement and intercalated disc structural abnormalities, characterized in that, A sgRNA targeting exon 6 of the zebrafish cyth3a gene was designed. The cyth3a gene in wild-type zebrafish embryos was mutated using a mixture of sgRNA and Cas9 protein. The resulting F0 generation zebrafish were crossed with wild-type zebrafish, and F1 generation heterozygotes were selected from the offspring. The F1 generation heterozygotes were then inbred, and positive homozygotes were selected from the offspring to form the F2 generation zebrafish, which is the zebrafish model of arrhythmia with atrial enlargement and intercalated disc structure abnormalities.

2. The method for constructing the arrhythmic zebrafish model with atrial enlargement and intercalated disc structural abnormalities according to claim 1, characterized in that, The mixture of sgRNA and Cas9 protein was introduced into wild-type zebrafish embryos by microinjection to obtain F0 generation zebrafish.

3. The method for constructing a zebrafish model of arrhythmia with atrial enlargement and intercalated disc structural abnormalities according to claim 1, characterized in that, The sgRNA / Cas9 protein mixture contained 1.5 µM sgRNA and 0.5 µg / µL Cas9 protein.

4. The application of the arrhythmic zebrafish model with atrial enlargement and intercalated disc structural abnormalities constructed by the construction method according to any one of claims 1-3 in screening drugs for the treatment of arrhythmic cardiomyopathy.

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