Efficient construction method and detection method of zebrafish heart failure model

Abstract

The application discloses a method for establishing and evaluating a zebrafish heart failure model and belongs to the technical field of experimental animal models. The method comprises the following steps: S1, preparing zebrafish embryos; S2, pretreating the embryos; S3, placing the pretreated embryos in exposure liquid containing isopropyl adrenaline for drug bath treatment to complete the construction of a heart failure model; S4, observing and evaluating the morphology of the embryos; and S5, detecting and analyzing the heart function, including measuring the heart rate, the ventricular diastolic / systolic end area and the long axis / short axis diameter, and calculating core parameters such as the area change fraction, the shortening fraction and the ejection fraction. According to the standardized isopropyl adrenaline induction scheme, the zebrafish is stably induced to exhibit typical heart failure phenotypes such as a slow heart rate, pericardial edema, ventricular dilation and venous stasis within 72 hours after fertilization, and the survival rate of the embryos is significantly reduced. The method has the advantages of a short modeling cycle, simple operation, good repeatability, and the ability to simultaneously complete the comprehensive evaluation of the heart structure and function at the whole animal level and the tissue level, and is suitable for high-throughput applications of cardiovascular drug screening and cardiac toxicity evaluation.

Description

Technical Field

[0001] This invention relates to the fields of biology and medicine, particularly to the field of experimental animal model technology, specifically a method for constructing and analyzing a zebrafish heart failure model. Background Technology

[0002] Heart failure is the end stage of many cardiovascular diseases, and its pathological mechanisms and treatment strategies rely heavily on stable and reliable animal models. While traditional mammalian models can mimic the human heart failure phenotype, they suffer from inherent drawbacks such as long modeling cycles, high costs, and phenotypic instability, hindering drug development efficiency. Zebrafish, as an important vertebrate model organism, possesses significant advantages such as embryonic transparency, in vitro development, and a short reproductive cycle. 84% of human disease-causing genes have corresponding genes in zebrafish, exhibiting similar pathophysiological characteristics. Zebrafish possess cardiovascular, nervous, and metabolic systems similar to mammals. In cardiovascular research, zebrafish and humans are remarkably similar in early organ development and pumping function, thus demonstrating unique application value. Thanks to the optical transparency of the embryo, changes in heart morphology and function can be monitored in real-time within living embryos. Furthermore, early zebrafish embryos obtain oxygen through diffusion rather than relying on the cardiovascular system, allowing the embryos to survive even with impaired cardiac function during the construction of heart failure models, providing a unique technical window for studying heart failure mechanisms and evaluating drugs. Zebrafish, as an ideal vertebrate model, have shown unique application value in the research of drugs for heart failure.

[0003] Isoproterenol (ISO) is a non-selective β-adrenergic receptor agonist that overactivates the Gs-AC-cAMP-PKA signaling axis. Overexcitation of this pathway leads to a surge in myocardial oxygen consumption, calcium overload, and mitochondrial energy metabolism collapse, ultimately triggering cardiomyocyte apoptosis. As a water-soluble reagent, ISO requires no organic solvents for dissolution. This physicochemical property allows it to be directly and uniformly dispersed in zebrafish embryo culture systems in aqueous solution, perfectly suited to the "drinking" administration method used in zebrafish models, while avoiding the unknown toxicity or physiological interference of solvents on zebrafish embryos. Because zebrafish embryos rely on diffuse oxygen supply in their early stages, their cardiovascular system is particularly sensitive to the relative ischemia and energy depletion induced by ISO.

[0004] Therefore, this application employs the ISO continuous medicated bath-induced chronic heart failure model in zebrafish juveniles, and systematically evaluates it using a combination of methods including heart rate measurement, cardiac morphology observation, and cardiac function parameter analysis. This model is characterized by its short establishment period, simple operation, quantifiable phenotype, and ability to simulate the neurohumoral activation and energy metabolism disorders characteristic of clinical chronic heart failure. Summary of the Invention

[0005] This invention provides a standardized method for constructing and evaluating the multidimensional function of a zebrafish heart failure model. By establishing a standardized ISO induction protocol and a multi-parameter dynamic evaluation system, this invention achieves quantifiable, highly reproducible, and scalable phenotypes in the zebrafish heart failure model. Within 72 hours of fertilization, the model stably exhibits typical pathological phenotypes such as bradycardia, pericardial edema, ventricular dilatation, and venous congestion, and significantly reduces embryo survival rate. Based on this, multidimensional cardiac function parameters allow for a comprehensive evaluation of cardiac structure and function simultaneously at both the whole animal and tissue levels. The model construction method includes the following steps: Preparation of S1 zebrafish embryos.

[0006] S2 is used to pretreat zebrafish embryos.

[0007] S3 Drug Treatment: The zebrafish embryos obtained in step S1 were exposed to an exposure solution to complete the construction of the zebrafish heart failure model.

[0008] S4 zebrafish embryo observation.

[0009] S5 cardiac function detection and analysis.

[0010] As one embodiment of the zebrafish heart failure model construction method described in this invention, the preparation in step S1 includes the following steps: Randomly select healthy male and female individuals of uniform size and reproductive capacity, and feed them brine shrimp four times the day before breeding. The night before breeding, transfer them to a spawning tank at a male-to-female ratio of 1:2, and turn off the light to allow them to enter a dark period. Turn on the light at 9:00 AM the following morning to induce spawning. After spawning, collect all fertilized eggs. Gently wash the collected embryos with E3 culture medium, and remove abnormal embryos such as those with broken shells, developmental arrest, or cytoplasmic aggregation. Place the selected healthy embryos in a culture dish for culture, changing the E3 culture medium every 12 hpf, and removing abnormal embryos such as those with broken shells, developmental arrest, or cytoplasmic aggregation.

[0011] As one embodiment of the zebrafish heart failure model construction method described in this invention, wild-type AB strain zebrafish (Danio Rerio) were selected. These wild-type (AB strain) adult zebrafish, purchased from the National Zebrafish Resource Center (Wuhan), had a weight of (0.5±0.1) g and a body length of (3.0±0.5) cm (mean ± standard error). The adult zebrafish were placed in aerated filtered water with dissolved oxygen levels of 5.6-7.2 mg / L and a pH maintained at 7.4±0.5. The ambient temperature was kept stable at 28±1℃, and the water temperature at 26±1℃. The light-dark cycle was maintained at 14h:10h. Live brine shrimp or zebrafish-specific feed were fed three times daily, with the aerated water changed daily. Uneaten feed and excrement were promptly removed to maintain water stability.

[0012] As one embodiment of the zebrafish heart failure model construction method of the present invention, the pretreatment in step S2 of the zebrafish includes the following steps: dissecting the 24 hpf zebrafish embryos to prevent the embryonic membrane from affecting the soaking of the drug. The dissection is performed using biological forceps under a microscope.

[0013] In one embodiment of the zebrafish heart failure model construction method of the present invention, the working fluid is changed daily.

[0014] As one embodiment of the zebrafish heart failure model construction method of the present invention, the preparation method of the E3 culture medium includes the following steps: weigh 2.94g sodium chloride, 0.13g potassium chloride, 3.3663g calcium chloride, and 0.396g magnesium sulfate, and dissolve them in 1L of pure water to prepare the solution.

[0015] As one embodiment of the zebrafish heart failure model described in this invention, isoproterenol solution is prepared by dissolving isoproterenol powder in E3 and used immediately after preparation.

[0016] As one embodiment of the analysis method described in this invention, the exposed embryo is fixed in a left lateral decubitus position, and a ventricular pulsation video is recorded for 15 seconds. The heart rate (HR) and ventricular pulsation video are analyzed using ViewPoint software, and relevant cardiac functions are calculated based on the end-diastolic area (EDA) and end-systolic area (ESA).

[0017] The beneficial effects of this invention are: 1. The modeling system of this invention has a high degree of standardization, is easy to operate, and produces stable and reliable phenotypes. The simple solution preparation and drug bath operation significantly reduce the complexity of experimental operations and batch-to-batch variability, ensuring high reproducibility of heart failure phenotype induction, and effectively overcoming the technical bottlenecks of traditional modeling methods such as cumbersome operation and unstable phenotypes.

[0018] 2. The method described in this invention has a short modeling cycle, is easy to operate, and has good stability and reproducibility. It is suitable for high-throughput applications in screening for the cardioprotective activity of drugs and assessing cardiovascular toxicity, providing reliable technical support for basic research and drug development in heart failure. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the morphological features of the zebrafish heart failure model in an embodiment of the present invention.

[0020] Figure 2 This is a bar chart of cardiac function indicators in a zebrafish heart failure model according to an embodiment of the present invention.

[0021] Figure 3 This is a bar chart of cardiac function indicators in a zebrafish heart failure model according to an embodiment of the present invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the experimental methods used in the embodiments and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0023] (1) Construction of a zebrafish heart failure model Wild-type AB-strain adult zebrafish were selected, with a weight of (0.5±0.1) g and a body length of (3.0±0.5) cm (mean ± standard error). Adult zebrafish were placed in aerated and filtered water with dissolved oxygen levels of 5.6-7.2 mg / L and a pH maintained at 7.4±0.5. The ambient temperature was kept stable at 28±1℃, and the water temperature at 26±1℃. The light-dark cycle was maintained at 14h:10h. Live brine shrimp or zebrafish-specific feed were fed three times daily, with the aerated water changed daily. Uneaten food and excrement were promptly removed to maintain water stability. For spawning, healthy males and females of uniform size and reproductive capacity were randomly selected. The fish were fed brine shrimp four times the day before breeding and transferred to the spawning tank the night before breeding at a female-to-male ratio of 1:2. The lights were then turned off to allow the fish to enter the dark period. The following morning at 9:00 AM, light was turned on to induce oviposition. After oviposition, fertilized eggs were collected. The collected embryos were gently washed with E3 culture medium, and abnormal embryos with broken shells, developmental arrest, or cytoplasmic aggregation were removed. The selected healthy embryos were then grouped and processed according to the experimental design protocol.

[0024] Zebrafish embryos were dissected at 24 hpf to prevent the embryonic membrane from affecting the drug immersion. Healthy dissected zebrafish were selected and randomly assigned to six-well plates, with 20 zebrafish juveniles per well. The control group received 4 mL of E3 culture medium per well. Isoproterenol powder was weighed and dissolved in E3 solution to prepare isoproterenol solutions with concentration gradients of 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, and 20 mg / mL. 2 mL of isoproterenol solution and 2 mL of E3 solution were added to each well. The final isoproterenol immersion solutions were prepared at concentrations of 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mg / mL. Three replicates were set up for each group. The six-well plates were placed in an incubator, and the heart rate of each group was recorded. The incubator temperature was maintained at 28±1℃, and the light-dark cycle was 14 h:10 h. During the culture period, the embryonic development process was observed daily at regular intervals, and dead individuals were promptly recorded and removed. Simultaneously, fresh E3 culture medium and the appropriate concentration of isoproterenol working solution were replaced daily to ensure stable processing conditions.

[0025] (2) Observation of zebrafish embryos and cardiac function testing Remove zebrafish fry from the incubator and place them at room temperature for 15 minutes to acclimatize and prevent stress from affecting the experiment. Record the mortality rate, pericardial edema rate, and deformity rate of the zebrafish fry. Use a Busper pipette to transfer the zebrafish fry to a clean glass slide. Place the slide under a stereomicroscope, adjust the zebrafish's position to a lateral recumbent position, adjust the focus, and start video recording. Stop recording after 15 seconds and record the number of heartbeats. Multiply the number of heartbeats recorded in 15 seconds by 4 to obtain the number of heartbeats per minute, which is the heart rate.

[0026] Import the captured video into Adobe Premiere Pro and play it frame by frame. Extract the ventricular systole and end-diastole frames from the same cardiac cycle and export them as JPG images. Import the images into Image Pro Plus and use the Measure tool to directly measure the diastolic ventricular long axis (Ld) and short axis (Dd), diastolic ventricular area (EDA), systolic ventricular long axis (Ls) and short axis (Ds), and systolic ventricular area (ESA). Then calculate the cardiac function parameters using the following formula: Area change FAC = 100 * (EDA - ESA) / EDA ventricular fractional shortening (FSS) = 100 * (Dd - Ds) / Dd ventricular long axis shortening fraction FSL = 100 * (Ld - Ls) / Ld End-diastolic volume (EDV) = 4 / 3 × π × (Ld / 2) × (Dd / 2)² End-contraction volume ESV = 4 / 3 × π × (Ls / 2) × (Ds / 2)2 Output per game SV=EDV-ESV Ejection fraction EF = 100 * SV / EDV The experimental results showed that, compared with the control group (containing only E3 culture medium), the zebrafish juveniles treated with isoproterenol exhibited typical morphological changes of heart failure, including pericardial edema, ventricular dilation, and venous sinus congestion (e.g., Figure 1 (As shown in the figure). Meanwhile, the mortality rate, pericardial edema rate, and malformation rate of embryos in the treatment group were significantly higher than those in the control group, and showed a concentration-dependent trend.

[0027] By recording and counting ventricular beats, the heart rate in the isoproterenol treatment group was significantly slower than that in the control group (e.g., ...). Figure 2As shown in the figure), this indicates that the drug induced significant bradycardia. Measurements of ventricular area and long and short axes at end-diastole and end-systole, along with calculated fractional area change (FAC), fractional short axis shortening (FSS), fractional long axis shortening (FSL), stroke volume (SV), and ejection fraction (EF), all showed significant reductions in the treatment group compared to the control group (e.g., ...). Figure 3 As shown in the figure, this indicates that the heart's contractile function and pumping ability are severely impaired.

[0028] Multiple independent experiments have shown that this modeling method has good consistency and reproducibility, and the heart failure phenotype induction rate is stable, making it suitable for subsequent drug screening and functional evaluation.

Claims

1. A method for constructing a zebrafish heart failure model, characterized in that, Includes the following steps: S1. Zebrafish embryo preparation: Collect healthy zebrafish fertilized eggs and culture them in E3 culture medium until the required developmental stage; S2. Embryo pretreatment: Zebrafish embryos that have developed to 24 hpf are dissected to remove the egg membrane; S3. Drug treatment: The embryos obtained in step S2 are placed in an exposure solution containing isoproterenol for drug bath culture. The exposure solution is prepared by dissolving isoproterenol in E3 culture medium, and the final concentration of isoproterenol is 0.02-10 mg / mL. S4. Embryo observation: During and after drug treatment, the embryo mortality rate, pericardial edema rate and malformation rate should be observed and recorded regularly. S5. Cardiac Function Testing and Analysis: Processed zebrafish juveniles were fixed in a lateral recumbent position, and ventricular pulsation videos were recorded to measure heart rate. Based on the end-diastolic area (EDA), end-systolic area (ESA), diastolic long axis (Ld) and short axis (Dd), and systolic long axis (Ls) and short axis (Ds), the fractional change in area (FAC), fractional shortening of the short axis (FSS), fractional shortening of the long axis (FSL), end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and ejection fraction (EF) were calculated to complete the cardiac function assessment.

2. The construction method according to claim 1, characterized in that, The zebrafish embryos described in step S1 were of wild-type AB strain, cultured at a temperature of 28±1℃, with a light-dark cycle of 14h:10h.

3. The construction method according to claim 1, characterized in that, In step S2, the membrane dissection is performed using biological forceps under a microscope, and abnormally developing or damaged embryos are removed after the membrane is dissected.

4. The construction method according to claim 1, characterized in that, The final concentration of isoproterenol in step S3 is preferably 0.2-2 mg / mL, more preferably 2 mg / mL.

5. The construction method according to claim 1, characterized in that, The medicated bath culture time in step S3 is 48-96 hours, with fresh exposure solution replaced daily, and the culture temperature is 28±1℃.

6. The construction method according to claim 1, characterized in that, The ventricular beat video recorded in step S5 is 15 seconds long. End-diastolic and end-systolic images are obtained by frame-by-frame analysis, and the area and diameter are measured using Image Pro Plus software.

7. An analytical method for a zebrafish heart failure model obtained by the construction method according to any one of claims 1-7, characterized in that, Includes the following steps: The model juvenile fish were fixed in the left lateral decubitus position, and videos of blood flow in the dorsal aorta were recorded to measure the average blood flow velocity and vessel diameter, and to calculate shear stress and blood flow. At the same time, videos of ventricular pulsation were recorded, and the heart rate was analyzed using ViewPoint software. Based on the end-diastolic and end-systolic images, the fractional area change, fractional shortening, stroke volume, ejection fraction, and cardiac output were calculated to comprehensively evaluate the degree of heart failure.

8. The analytical method according to claim 7, characterized in that, The video of dorsal aortic blood flow must be at least 10 seconds long, the video of ventricular pulsation must be at least 15 seconds long, and the frame rate must be at least 120fps.

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