Construction method and application of a zebrafish model of arrhythmic cardiomyopathy

Abstract

The invention belongs to the technical field of animal model construction, and relates to a construction method and application of an arrhythmic cardiomyopathy zebrafish model. An sgRNA targeting exon 2 of a zebrafish brd4 gene is designed, a mixture of sgRNA and Cas9 protein is used to mutate the brd4 gene of a wild-type zebrafish fertilized egg, the obtained F0 generation zebrafish is mated with a wild-type zebrafish, and positive heterozygous F1 generation zebrafish are screened out from the born zebrafish, which is an arrhythmic cardiomyopathy zebrafish model; through cardiac pathology, cardiac function detection, heart rate and swimming test indicators, it is confirmed that the brd4 heterozygous mutant zebrafish can develop arrhythmic cardiomyopathy. The model construction method is simple, efficient and has a high success rate, and provides great help and convenience for evaluating or screening drug research for treating arrhythmic cardiomyopathy.

Description

Technical field:

[0001] The invention belongs to the technical field of animal model construction, and specifically relates to a construction method and application of a zebrafish model of arrhythmic cardiomyopathy. The model can be used to screen drugs for treating arrhythmic cardiomyopathy. Technical background:

[0002] Arrhythmic cardiomyopathy (ACM) is the latest term for myocardial disease, originally called arrhythmic right ventricular dysplasia / cardiomyopathy, which manifests as malignant ventricular arrhythmias and fibrofatty infiltration of the biventricular myocardium, with right ventricular involvement being more common. The prevalence of ACM in the general population is estimated to be 1 in 2,000 to 5,000 cases, depending on the patient cohort studied. Although considered a rare heart disease, ACM with sustained ventricular arrhythmias is a major risk condition for a large proportion of sudden cardiac death, especially in young people and athletes. More than 25 gene mutations have been identified that can cause ACM, accounting for 60% of ACM cases. For patients with known genetic causes, the molecular mechanisms of ACM disease progression remain poorly understood. Currently, the clinical management of ACM patients focuses on general control of heart failure and prevention of arrhythmic sudden cardiac death, and there is no specific treatment for ACM. Therefore, the establishment of an ACM model is conducive to studying its pathogenic mechanism and also provides a drug screening model for clinical drug use.

[0003] Traditionally, mammals such as rodents have been the gold standard for animal models when studying disease pathophysiology and potential disease mechanisms of action, as their physiology is well conserved compared to humans. Rodent models are limited in their ability to functionally validate large numbers of genetic variants and discover new genetic disease associations at scale, due to their relatively long experimental procedures and high costs, resulting in low throughput. Another inherent limitation when using mice to model arrhythmic diseases such as ACM is that their average heart rate is approximately 500 beats per minute, which is much higher than the average human heart rate of approximately 60-90 beats / min. In contrast, zebrafish have an average heart rate of approximately 100 beats per minute and share very similar cardiac electrophysiological characteristics with humans. Zebrafish are a small tropical vertebrate that shares 87% genetic similarity with humans. Their embryonic transparency, high fecundity, and conserved cardiac electrophysiology compared to humans make them a popular animal model for studying cardiovascular system development and cardiac function, as well as for screening cardiotoxic drugs. However, to date, no method has been found to construct a zebrafish model of arrhythmic cardiomyopathy. Summary of the invention:

[0004] The purpose of this application is to overcome the shortcomings of the existing technology and provide a method for constructing a zebrafish model of arrhythmic cardiomyopathy and its application.

[0005] The present application provides a method for constructing a zebrafish model of arrhythmic cardiomyopathy, comprising:

[0006] (1) preparing an sgRNA targeting exon 2 of the zebrafish brd4 gene, wherein the sequence of the sgRNA is as shown in SEQ ID NO: 1, and the sequence is 5'-GGATTCGGGTTGAAGGAAGA-3';

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

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

[0009] (4) The F0 generation zebrafish is mated with wild-type zebrafish, and positive heterozygous F1 generation zebrafish are screened out from the born zebrafish, which is the arrhythmic cardiomyopathy zebrafish model.

[0010] In one embodiment, the Cas9 / sgRNA mixture is introduced into wild-type zebrafish embryos by microinjection.

[0011] In one embodiment, PCR and Sanger sequencing were used to screen out positive heterozygous F1 generation zebrafish, and the PCR detection primers used were F: 5'-CGAGGAGTGCAATGGAAT-3' (SEQ ID NO: 2), R: 5'-CTCTTCAGCACCACCTTC-3' (SEQ ID NO: 3), and the amplified target fragment was 294 bp.

[0012] The genotype of the F1 generation zebrafish in the present invention is determined by extracting fish tail DNA, using PCR amplification and Sanger sequencing.

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

[0014] The present invention also provides application of the arrhythmic cardiomyopathy zebrafish model in screening drugs for treating arrhythmic cardiomyopathy.

[0015] The pathological characteristics of the brd4 gene mutant zebrafish of the present invention are: compared with the wild type, the heart rate of the brd4 gene mutant zebrafish in the embryonic period (5 days after fertilization) is slowed down by about 152 times / min to 134 times / min, with irregular heartbeat, pericardial edema, heart malformation, and a decrease in cardiac contraction fraction (FS) of 22%, about 15%. The heart rate of the 6-month-old brd4 gene mutant zebrafish is slowed down to 88 times / min; the heart pumping function is weakened, the cardiac ejection fraction (EF) is about 41%, the contraction fraction (FS) is about 14%, the stroke volume (SV) is about 0.4μL, and the ventricular area change fraction (FAC) is about 30%; the heart rhythm is irregular, sinus arrest occurs, and the QT wavelength increases; the density of myocardial cells increases, the myocardial cells are hypertrophic, and the arrangement of myocardial nodules is disordered; the desmosomes at the junction of myocardial cells are reduced and the density decreases. In addition, the swimming ability of the brd4 gene mutant zebrafish decreases, and some fish suffer from heart failure after swimming, leading to death.

[0016] The method for constructing a brd4 gene mutant zebrafish model provided by the present invention is to construct a brd4 mutant zebrafish model through the CRISPR / Cas9 system; however, other conventional gene editing methods can also be used to obtain the zebrafish brd4 gene mutant.

[0017] Compared with the prior art, the present invention constructs a zebrafish model of arrhythmic cardiomyopathy for the first time. The model construction method is simple, efficient and has a high success rate. The model can be used for research on the regulation of cardiac function by the brd4 gene. It can also be used as an animal model of ACM for drug screening and other purposes, providing great help and convenience for evaluating or screening drugs for treating arrhythmic cardiomyopathy. It can also provide toxicological research for drug screening targeting the brd4 gene for the treatment of cancer or heart disease. Description of the drawings:

[0018] Figure 1 This is the effective Sanger sequencing map of the F0 generation mutant zebrafish of the present invention.

[0019] Figure 2 The present invention is a schematic diagram of the experimental results of mutating the brd4 gene of zebrafish fertilized eggs, wherein A is a schematic diagram of the brd4 mutant allele generated by CRISPR / Cas9, the dotted line indicates the deleted nucleotide, the sgRNA sequence is indicated in green, and the PAM (protospacer adjacent motif) site is indicated by a red underline; B is a wild-type brd4, a heterozygous mutant brd4 + / - Sequencing results of the sequence, the framed sequence represents the region containing the site in the wild-type sequence that is deleted in the mutant; C is the BRD4 protein and the truncated protein after mutation.

[0020] Figure 3 Schematic diagram of the cardiac function test results of mutant zebrafish embryos of the present invention, wherein A is a wild-type zebrafish embryo (WT) and a brd4 heterozygous mutant embryo (brd4 + / - ) White light phenotype 5 days after fertilization; B is the comparison of heart rate (Bmp) between wild type and mutant; C is the comparison of cardiac fractional shortening (FS) between wild type and mutant.

[0021] Figure 4 The present invention is a schematic diagram of the comparative results of cardiac ultrasound detection of adult zebrafish mutants and wild-type adult fish, wherein A is the heart rate of adult fish; B is the echocardiogram of the heart during systole (left figure) and diastole (right figure); C is the index of cardiac pumping function; D is the electrocardiogram; and E is the statistics of QTc interval, PR interval, QR interval and QRS interval.

[0022] Figure 5 Schematic diagram of swimming test results of wild-type and mutant zebrafish involved in the present invention, wherein A is the mutant brd4 at 6 months + / - Compared with the maximum swimming speed of wild type; B is the survival curve after swimming.

[0023] Figure 6 The 6-month-old brd4 + / - Representative images of isolated hearts of mutants and wild-type (WT) controls and quantification of ventricular surface area (VSA) normalized to body weight (BW / g), n = 5 fish per group; A is the heart image, and B is the quantification of ventricular surface area (VSA) normalized to body weight (BW / g).

[0024] Figure 7 Schematic diagram of the comparison of cardiac histological detection results between mutant zebrafish and wild-type adult fish involved in the present invention, wherein A is brd4 at 6 months + / - Representative images of H&E staining of the apical region of mutants and wild-type controls and quantification of myocardial density; B is fluorescent immunostaining of sarcomeres with anti-α-actin antibody (green); C is TEM image confirming the expression of brd4 at 6 months of age. + / - The mutant heart showed myofiber degeneration (yellow asterisk) and abnormal mitochondrial swelling (green arrow) (20 μm×); D is abnormal cell junction (200 nm×). Specific implementation method:

[0025] The technical solution of the present invention is described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0026] Embodiment 1:

[0027] This embodiment relates to a method for constructing a zebrafish model of arrhythmic cardiomyopathy, which is obtained by constructing a zebrafish brd4 gene mutant. The specific steps are as follows:

[0028] (1) Taking the zebrafish brd4 gene as the target sequence, the target site sgRNA sequence was designed and synthesized on exon 2. The sgRNA was synthesized by GenScript Biotechnology Co., Ltd.; the DNA sequence of the sgRNA was 5'-GGATTCGGGTTGAAGGAAGA-3' (SEQ ID NO: 1);

[0029] Among them, the zebrafish brd4 gene (ZDB-GENE-030131-267), the public NCBI transcript NM_001111281.1 encodes 1444 amino acids.

[0030] (2) Preparation of injection needle: Before microinjection, use a needle puller to make a 1 mm capillary into a needle for microinjection. Be extremely careful when making the microinjection needle to prevent the needle tip from breaking. Mix sgRNA and Cas9 protein (final concentration of sgRNA is 1.5 uM, final concentration of Cas9 protein is 0.5 ug / uL), incubate at 37°C for 10 min, and after obtaining the Cas9 / sgRNA mixture, add phenol red indicator and inject it into the microinjection needle using an eppendorf long pipette tip to avoid bubbles in the injection needle. (3) Microinjection: Use an embryo injection instrument to inject the sgRNA and Cas9 protein mixture into wild-type zebrafish 1-cell embryos for CRISPR / Cas9 gene editing. At least 200 embryos should be injected. Place the embryos injected with the sgRNA and Cas9 protein mixture in an embryo culture dish and add embryo culture medium (0.292 g / L NaCl, 0.013 g / L KCl, 0.044 g / L CaCl2, 0.081 g / L MgSO4, pH 7.2); some uninjected embryos from the same batch were kept as wild-type controls; all embryos were cultured in a 28.5°C incubator;

[0031] (4) Identification of whether sgRNA is successful: After the injection is completed and the fertilized eggs develop to 3 days, 3 embryos from the injection group and the non-injection group (wild type) are randomly taken and placed in EP tubes. First, 40 μL of 50 mM NaOH is added and lysed in a PCR instrument at 95°C for 30 minutes; then briefly vortex and centrifuge for 10 seconds, and 4 μL of Tris-HCl (pH = 8.0) is added to the lysate for neutralization, vortexed for 30 seconds, and the supernatant after centrifugation is the genomic DNA of the zebrafish embryo; using it as a template, PCR amplification is performed using the upstream and downstream detection primers of the target sequence (F: 5'-CGAGGAGTGCAATGGAAT-3' (SEQ ID NO: 2), R: 5'-CTCTTCAGCACCACCTTC-3' (SEQ ID NO: 3)). The PCR reaction system and reaction conditions are shown in Tables 1 and 2). After the PCR product is subjected to nucleic acid electrophoresis and the band is bright and free of mixed bands (the amplified target fragment is 294bp), it is sent to a sequencing company for Sanger sequencing. If the sequencing result is a single peak at the target site, no mutation has occurred; if the result is a random peak 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 shown.

[0032] Table 1 PCR amplification reaction system

[0033]

[0034] Table 2 PCR reaction conditions

[0035]

[0036] Identify the mutation of the brd4 gene in the F0 generation zebrafish: At this time, it can be read in the sequencing results that the F0 generation mutant zebrafish has a base deletion compared to the wild-type zebrafish.

[0037] (5) The F0 generation zebrafish were hybridized with wild-type zebrafish to produce F1 generation zebrafish, which were raised to adulthood. DNA was extracted by cutting the tail, and the target band was amplified by PCR to verify the base mutation. Sanger sequencing was used to verify that the heterozygous mutant zebrafish had a 4-base deletion compared with the wild-type zebrafish ( Figure 2 The stably inherited heterozygotes in F1 were selected as zebrafish models of arrhythmic cardiomyopathy and were cultured for subsequent experiments.

[0038] F1 generation brd4 + / -When heterozygous mutant zebrafish are self-pollinated, homozygous mutant zebrafish can be screened during the embryonic period, but they die in the early development or late growth process and no adult homozygous zebrafish can be obtained. Therefore, the disease model is preserved and maintained with zebrafish with brd4 heterozygous mutation.

[0039] Embodiment 2:

[0040] This embodiment involves an experiment to analyze the effect of brd4 gene mutation on zebrafish cardiac function, including:

[0041] (1) Embryonic cardiac function: brd4 + / - Heterozygous zebrafish were inbred to obtain brd4 mutant embryos. After culturing in a 28.5°C incubator for 5 days, phenotype observation was performed and heart rate and cardiac ejection fraction were measured. Specifically, the embryos were taken out with a pipette and placed in 0.02% tricaine for anesthesia for 1 min, and placed sideways on 3% methylcellulose. The beating heart was recorded using a Zeiss Axioplan 2 differential interferometer camera lens with 20x magnification, and the heart rate and shortening fraction FS (%) were calculated using video clips = (diastolic width - systolic width) / diastolic width * 100; wild-type zebrafish embryos were used as controls. The results are shown in Figure 3 shown.

[0042] from Figure 3 A White light phenotype shows that brd4 mutants have pericardial edema and heart malformations. Figure 3 B shows that during the embryonic period (5 days after fertilization), the heart rate of the brd4 mutant was 134 beats / min, which decreased to about 152 beats / min compared with the wild type, indicating an irregular heart rate. Figure 3 C shows that the fractional shortening (FS) of the heart of the brd4 mutant is about 15%, which is reduced to 22% compared with that of the wild type.

[0043] (2) Cardiac function of adult fish: When brd4 mutant zebrafish and wild-type zebrafish grow to 6 months old, their cardiac function, heart rate and maximum swimming speed are tested using the Vevo3100 high-frequency imaging system, iWorx system and swimming tunnel system; and their cardiac changes are analyzed using pathological histological techniques. The specific steps are as follows:

[0044] 1) In vivo echocardiography of adult fish hearts: A 50MHz (MX700) Vevo 3100 high-frequency imaging system was used to measure and analyze cardiac function indicators in adult zebrafish. Acoustic gel was applied to the probe surface to provide adequate coupling with the tissue interface. Six-month-old adult zebrafish were anesthetized with 0.02% tricaine for 5 minutes and placed in a sponge with the abdomen facing up. A 50MHz (MX700) transducer was placed above the zebrafish to provide sagittal imaging of the heart. B-mode images used an axial imaging field of view of 8.00mm, a lateral imaging field of view of 8.73mm, a frame rate of 152Hz, a medium persistence, and a transmission focus at the center of the heart. Image quantification was performed using the data package in the VevoLAB workstation, and ventricular dimensions were measured based on B-ultrasound images using the following three indicators: ejection fraction, shortening fraction, and ventricular area change fraction; the results are shown in Figure 2. Figure 4 As shown in A-4C.

[0045] from Figure 4 As can be seen in A-4C, the heart rate of a 6-month-old wild-type zebrafish is about 116 beats / min, brd4 + / - The heart rate of heterozygous mutant zebrafish slowed down to 88 beats / min; the cardiac ejection fraction (EF), fractional shortening (FS), stroke volume (SV) and fractional area change (FAC) of wild-type zebrafish were approximately 53%, 23%, 0.55 μL and 37%, respectively, while brd4 + / - The EF, FS, SV, and FAC of mutant zebrafish were approximately 41%, 14%, 0.4 μL, and 30%, respectively, indicating that compared with wild-type zebrafish, brd4 + / - The mutant zebrafish's hearts have reduced pumping function.

[0046] 2) ECG: One week before the ECG test, the fish underwent microsurgery under a stereoscopic dissecting microscope to remove the silver epithelial layer outside the heart. After one week of recovery from microsurgery, the fish were anesthetized with 0.02% tricaine for 5 minutes, and then the ECG was recorded for 4 minutes using the iWorx system. The results are shown in Figure 4 D and 4E.

[0047] from Figure 4 D and 4E show that brd4 + / - The zebrafish mutant showed sinus arrest (SA) and a longer QT wavelength, indicating that the constructed brd4 + / - The mutant zebrafish showed irregular heartbeats and slow heart rates.

[0048] 3) Swimming tunnel test: brd4 + / -Mutant zebrafish were raised with age-matched wild-type zebrafish, and all fish were fasted for 24 h before the swimming ability was measured; to evaluate the swimming ability of adult fish, 6-month-old zebrafish were placed in a swimming tunnel with an initial water speed of 9 cm / s for a 20-min acclimatization period, and then the water flow was 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) of each fish were recorded. The critical swimming ability (Ucrit) is calculated as: Ucrit = Uii + [Ui × (Ti / Tii)], and then Ucrit is normalized to the body length (BL) of the corresponding individual. The results are shown in Figure 5 shown.

[0049] from Figure 5 It can be seen that brd4 + / - The critical swimming ability of mutant zebrafish is reduced ( Figure 5 A), after swimming, some fish suffered from heart failure and died, and the survival rate was less than 40%, which was significantly different from the wild type group ( Figure 5 B); Description brd4 + / - Mutant zebrafish have reduced motor abilities, and excessive exercise can easily lead to heart failure and death.

[0050] 4) Measurement of the ratio of ventricular surface area to body weight: Six-month-old zebrafish were anesthetized and weighed (BW), and then the hearts of individual zebrafish were dissected and imaged on a Leica stereo microscope with a millimeter ruler to measure the ventricular surface area (VSA). Figure 6 shown.

[0051] from Figure 6 It can be seen that brd4 + / - The mutant zebrafish did not show ventricular dilation and fat adhesion, indicating that brd4 + / Mutants - The cardiac phenotypes characteristic of arrhythmic zebrafish are no different from those of wild-type zebrafish.

[0052] 5) Histology: The hearts of 6-month-old zebrafish were immediately fixed with 4% formaldehyde fixative, embedded in paraffin, and stained with hematoxylin and eosin (H&E) after euthanasia. The Ni-U imaging system (Nikon) was used to capture images of the apex area, and ImageJ software was used to quantitatively determine the trabecular muscle density. Frozen section processing: The hearts of 6-month-old zebrafish were taken after euthanasia. The hearts were fixed with 4% paraformaldehyde fixative for 10 minutes, and then placed in a 30% sucrose aqueous solution at 4°C for dehydration for 24-48 hours until the tissue was completely precipitated. Finally, they were transferred to OCT embedding medium and stored at -80°C. The prepared samples can be directly sliced ​​at a later time using a freezing microtome (Leica CM1950). Anti-α-actin (1:120, Sigma-Aldrich, A7811) was used to stain the arrangement of myometrium. The results are as follows. Figure 7 A and 7B.

[0053] from Figure 7 A shows that at 6 months, brd4 + / - H&E staining of the apex region of the mutant showed a significant increase in the density of sarcomeres compared to the wild-type control group; Figure 7 B found brd4 + / - Immunofluorescence staining of the mutant hearts with anti-α-actin antibodies showed disruption of the sarcomere structure. + / - Myocardial fibers in mutant zebrafish undergo degeneration.

[0054] 6) Transmission electron microscopy (TEM): In the TEM study, the zebrafish hearts were immediately fixed in a fixative solution (4% paraformaldehyde and 1% glutaraldehyde in 0.1M pH 7.2 phosphate buffer) at room temperature for 1 h and then placed at 4°C overnight. The fixed samples were then processed and imaged using a HT7800 transmission electron microscope by Wuhan Saike Biotechnology Co., Ltd. Figure 7 C and 7D.

[0055] from Figure 7 C confirmed that at 6 months, brd4 + / - The cardiac muscle fibers of mutant zebrafish show degeneration phenotype (yellow asterisks) and abnormal mitochondrial swelling (green arrows); Figure 7 D shows the magnified cell junction, where the number of desmosomes and the density of intermediate filaments are reduced. These results indicate that brd4 + / - The mutants showed abnormalities in cardiac muscle fibers and cell junctions, which affected heart rate, indicating that the mutant zebrafish had arrhythmias.

[0056] The above results show that F1 generation brd4 + / - Heterozygous mutant zebrafish develop arrhythmic cardiomyopathy (ACM).

[0057] Embodiment 3:

[0058] This embodiment involves brd4 + / - Applications of mutant zebrafish, specifically:

[0059] The F1 generation brd4 gene mutant zebrafish constructed in Example 1 is used to screen drugs for treating ACM disease. The drug that can improve or rescue the phenotypes of brd4 mutant zebrafish fry, such as death, arrhythmia, morphological abnormalities, etc., is the target drug.

[0060] A wild-type control group, a brd4 mutant control group, a wild-type drug-treated group, and a brd4 + / - For the mutant drug treatment group, a 6-well cell plate was used, with 20 zebrafish embryos placed in each well, and cultured at 28.5°C. A 50 μM concentration of propranolol solution was prepared using zebrafish culture water. The drug was administered at a final concentration of 50 μM when the zebrafish embryos were 1 day old, and brd4 was observed according to the method of using the embryonic heart in Example 2. + / - Phenotypic changes in mutant zebrafish morphology, survival, heartbeat, etc. were recorded for a total of 12 days.

[0061] The results showed that under the treatment of 50 μM propranolol solution, brd4 + / - The time when the mutant zebrafish showed death symptoms was significantly delayed, indicating that the propranolol solution had a certain alleviating effect on the symptoms of the zebrafish model of the present invention, and propranolol could be used as a drug for treating ACM disease.

Claims

1. A method for constructing a zebrafish model of arrhythmic cardiomyopathy, characterized in that: An sgRNA targeting exon 2 of the zebrafish brd4 gene was designed, and the brd4 gene of the wild-type zebrafish fertilized eggs was mutated using a mixture of sgRNA and Cas9 protein. The obtained F0 generation zebrafish were mated with wild-type zebrafish, and positive heterozygous F1 generation zebrafish were screened from the born zebrafish, which was a zebrafish model of arrhythmic cardiomyopathy; the nucleotide sequence of the sgRNA was 5'-GGATTCGGGTTGAAGGAAGA-3'.

2. The method for constructing a zebrafish model of arrhythmic cardiomyopathy according to claim 1, characterized in that: The sgRNA and Cas9 protein mixture is introduced into wild-type zebrafish embryos by microinjection to obtain F0 generation zebrafish.

3. The method for constructing a zebrafish model of arrhythmic cardiomyopathy according to claim 1, characterized in that: The sgRNA concentration in the sgRNA and Cas9 protein mixture was 1.5uM, and the Cas9 protein concentration was 0.5ug / ul.

4. Use of the arrhythmic cardiomyopathy zebrafish model constructed by the construction method according to any one of claims 1 to 3 in screening drugs for treating arrhythmic cardiomyopathy.

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