Zebra fish heart electrophysiology detection method and application thereof

Abstract

The invention discloses a zebra fish heart electrophysiology detection method and application thereof, and belongs to the technical field of biological tissue dielectric property analysis. The method is mainly carried out by using a Taiwan solution especially suitable for keeping the vitality of the zebra fish heart and a dye solution special for the isolated heart of the zebra fish. The Taiwan solution is an isotonic ion solution, and various ions dissolved in the Taiwan solution can ensure ion exchange of the zebra fish heart, so that the zebra fish heart is prevented from being inactivated due to over-infiltration or water loss conditions and the like. The dye liquor prepared on the basis of the Taiwan's solution can ensure the activity of the zebra fish heart in the long-time dyeing process. The dye liquor is a combination of low-concentration and high-concentration calcium-sensitive infection solutions, and the use sequence is that the high concentration is performed firstly and then the low concentration is performed. The method can meet the requirements of various data required in electrophysiology related literatures, especially can reflect the change condition of calcium signals in cells, and also has the advantages of low cost, short experimental period and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biological tissue dielectric property analysis, and in particular relates to a zebrafish heart electrophysiological detection method and an application thereof in zebrafish heart calcium signal detection. Background Art

[0002] Electrophysiological indicators are often used in clinical medical diagnosis. For example, researchers have performed in vivo calcium imaging and electrophysiological recordings on early-stage zebrafish and found that Müller glial cells participate in and regulate spontaneous retinal activity waves through different receptors and transporters (Glial cells participate in regulating spontaneous retinal activity waves - Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences (Category 4 institution)). In addition, there are studies that use transparent skin to perform in vivo electrophysiological or optical imaging recordings of neuronal activity on a whole-brain scale (Technical Tips | In-depth analysis of research progress and methods of zebrafish visual neural signals). The electrophysiological detection technology currently used in zebrafish is still based on in vivo or ex vivo adult zebrafish hearts. There are few reports on electrophysiological detection technologies such as calcium imaging using ex vivo hearts of early zebrafish embryos, and whether this method can solve the above-mentioned defects of in vivo experiments is unknown in this field. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a zebrafish heart electrophysiological detection method and its application. The present invention is mainly carried out by using a Tyrode's solution that is particularly suitable for maintaining zebrafish heart vitality and a dye solution specifically used for zebrafish isolated heart. The Tyrode's solution is an isotonic ion solution, in which various ions dissolved can ensure the ion exchange of the zebrafish heart, and then keep it from being inactivated due to hyperosmosis or dehydration. The dye solution made based on the Tyrode's solution can ensure the zebrafish heart vitality during a long dyeing process. The dye solution is a combination of low and high concentration calcium-sensitive infection solutions, and the order of use is high concentration first, then low concentration. The present invention can meet the requirements of various data required in electrophysiology-related literature, especially can reflect the changes in calcium signals in cells, and because the heart of young zebrafish is small in size, the required calcium-sensitive dye is also very small, and there is no need to wait until it grows up to carry out experiments, so the present invention also has the advantages of low cost and short experimental cycle.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] One of the purposes of the present invention is to provide a Tyrode's solution capable of maintaining the activity of zebrafish isolated heart, wherein the Tyrode's solution is composed of 130-150 mM NaCl, 4-6 mM KCl, 0.5-1.5 mM MgCl2·6H2O, 5-10 mM HEPES, 5-10 mM D-glucose, 1.5-2.0 mM CaCl2·2H2O and 0-0.5 mM Na2HPO4·2H2O.

[0006] Preferably, the Tyrode's solution consists of 136 mM NaCl, 5.4 mM KCl, 1 mM MgCl2·6H2O, 10 mM HEPES (Sangon Biotech, CAS: [75277-39-3]), 5 mM D-glucose (Sangon Biotech, CAS# [50-99-7]), 1.8 mM CaCl2·2H2O and 0.3 mM Na2HPO4·2H2O.

[0007] The second object of the present invention is to provide a zebrafish isolated heart staining solution based on the Tyrode's solution, which is a combination of a low-concentration calcium-sensitive infection solution and a high-concentration calcium-sensitive infection solution.

[0008] Furthermore, the low-concentration calcium-sensitive infection solution of each system includes 6-8 μM Rhod2 and 9-11 μM Lebbistatin, and the high-concentration calcium-sensitive infection solution of each system includes 13-14 μM Rhod2 and 9-11 μM Lebbistatin, and the rest is made up by the Tyrode's solution; and when the low-concentration calcium-sensitive infection solution and the high-concentration calcium-sensitive infection solution are used, 1-3 μl F127 needs to be added to each 1 ml of the system.

[0009] Preferably, the low-concentration calcium-sensitive infection solution: 1 system = Rhod2 (7 μM) + Blebbistatin (10 μM) + Tyrode's solution to make up; the high-concentration calcium-sensitive infection solution: 1 system = Rhod2 (14 μM) + Blebbistatin (10 μM) + Tyrode's solution to make up; and when the low-concentration calcium-sensitive infection solution and the high-concentration calcium-sensitive infection solution are used specifically, 2 μl of F127 needs to be added to each 1 mL of the system.

[0010] The third object of the present invention is to provide a zebrafish isolated heart cardioplegia solution based on the Tyrode's solution, wherein each zebrafish isolated heart cardioplegia solution includes 9 to 11 μM Blebbistatin, and the rest is made up by the Tyrode's solution.

[0011] Preferably, the cardioplegic solution: 1 system = Blebbistatin (10 μM) + Tyrode's solution to complete.

[0012] The specific preparation of the zebrafish isolated heart dye solution and the zebrafish isolated heart cardioplegia solution also includes the step of reagent preparation: first, Rhod2 (Abcam ab142780) (calcium-sensitive dye) and Blebbistatin (Macklin B873251-5mg) (cardioplegia) stock solutions are prepared with DMSO, and F127 (invitrogen P3000MP) (punched) can be used directly.

[0013] A fourth object of the present invention is to provide a method for detecting zebrafish heart electrophysiology, which comprises using the zebrafish isolated heart dye solution and / or the zebrafish isolated heart arrest solution.

[0014] Furthermore, the zebrafish cardiac electrophysiological detection method comprises the following steps:

[0015] (1) Select juvenile zebrafish and isolate the intact heart by hypothermic shock;

[0016] (2) using the high concentration calcium-sensitive infection solution to immerse the heart for staining;

[0017] (3) removing the high-concentration calcium-sensitive infection solution and using the low-concentration calcium-sensitive infection solution to immerse the heart for staining;

[0018] (4) removing the low-concentration calcium-sensitive infection solution and submerging the heart with the cardioplegia solution;

[0019] (5) Computer records.

[0020] Furthermore, in step (1), the earliest 3dpf zebrafish can be used, and the heart is separated using an acupuncture needle, which is processed by reinforcing the needle body with tape to expose a little of the needle tip. The specific separation steps are as follows:

[0021] 1. Cut off the inflow and outflow tracts of the heart to separate the pericardial cavity;

[0022] 2. Separate the residual tissue fragments in the pericardial cavity to obtain a complete heart.

[0023] Furthermore, the staining method in step (2) is to slowly shake on a shaker for 1 to 3 minutes.

[0024] Preferably, the staining method is to slowly shake on a shaker for 2 minutes.

[0025] Furthermore, the staining method in step (3) is to slowly shake on a shaker for 8 to 12 minutes.

[0026] Preferably, the staining method is to slowly shake on a shaker for 10 minutes.

[0027] A fifth object of the present invention is to provide an application of the Tyrode's solution, the zebrafish isolated heart dye solution, the zebrafish isolated heart cardioplegia solution and / or the zebrafish heart electrophysiological detection method in zebrafish heart calcium signal detection.

[0028] The key of the present invention is:

[0029] First, in vitro cell electrophysiological experiments mainly record electrophysiological records of single cells or cell groups, such as neurons, cardiomyocytes, etc., while general large-scale isolated cardiac electrophysiological experiments use intact cardiac tissue for electrophysiological research, usually using Langendorff perfusion system, and perfusing nutrient solution through the coronary artery to maintain the physiological state of the heart. However, a small heart like zebrafish cannot be perfused to maintain cardiac vitality, but is kept alive by soaking in Tyrode's solution. This method is more simplified and can also monitor various indicators such as cardiac contraction and relaxation in real time, and can also observe cardiac changes caused by different drugs, and has strong controllability. Therefore, the present invention adopts the method of soaking in Tyrode's solution.

[0030] Second, the isolated heart is easy to inactivate, so the separation of the heart needs to be completed in a prepared Tyrode's solution. At the same time, different animal cells can withstand different osmotic pressures, so there are differences in the ion concentration of Tyrode's solution. The present invention has developed a Tyrode's solution that is particularly suitable for maintaining the vitality of the zebrafish heart. The Tyrode's solution is an isotonic ion solution, in which various ions are dissolved, which can ensure the ion exchange of the zebrafish heart, and then keep the zebrafish heart from inactivating due to hyperosmosis or dehydration, and can maintain the vitality of the zebrafish heart for a period of time; the glucose contained therein can provide nutrition for the zebrafish heart. At the same time, the dye solvent in the present invention is a mixed solution of the Tyrode's solution and the dye, and this mixed solution can ensure the vitality of the zebrafish heart during a long dyeing process.

[0031] 3. Generally, the operation that can be performed when electrophysiological techniques are applied to the heart in vivo is electrode stimulation, which is commonly used to measure electrocardiograms (ECG), and the operation is usually applied to adult fish. After the heart is operated in vitro, not only can electrode stimulation be performed (the larger heart of adult fish can be stimulated with electrodes, and the embryonic fish can use field stimulation because the heart is small and there is no electrode stimulation of suitable size), but also more operations can be performed, such as delivering the liquid medicine while observing the real-time transformation of calcium signals. In particular, the heart staining of embryonic fish can be carried out in the early stages of embryonic development. For example, in the drug screening process, if you want to pay attention to the influence of drugs on calcium signal changes, you can use the in vitro heart staining to pay attention to calcium signal indicators after collecting embryos for drug treatment. For example, calcium-related genes are knocked out in gene editing, and embryos can also be collected in F1 generation to observe whether there is a difference in calcium signal comparison with the normal group. Therefore, the in vitro separation technology of small hearts such as embryonic fish and juvenile fish is more valuable for research and more suitable for the present invention.

[0032] Fourth, it is difficult to separate the heart of a small fish, so the present invention uses an acupuncture needle. Specifically, the needle body of the acupuncture needle is reinforced with tape to expose a little of the needle tip. Only a needle as thick as an acupuncture needle can accurately peel the heart under a microscope. Although the electrophysiology of adult fish is more convenient, zebrafish, as a model organism used in high-throughput and short-cycle, is more often used for experiments with young fish. This is because the cardiac electrophysiological characteristics of young zebrafish are similar to those of humans, and the transparency of early zebrafish embryos allows direct observation of the effects of drugs on heart rhythm and arrhythmia, which is very useful for studying heart disease.

[0033] 5. There are many choices of calcium-sensitive dyes on the market. The present invention has verified in electrophysiological experiments on mice and rats that a single suitable concentration of the calcium-sensitive dye Rhod2 has a good effect, but a single concentration is not effective when applied to zebrafish. It may be that zebrafish are cold-blooded animals, or although they are both model animals, there are some differences in body structure and heart structure. Therefore, the present invention ultimately gives priority to dyeing with the above-mentioned high and low concentrations of calcium-sensitive infection solution in zebrafish cardiac electrophysiology, and then records the data. Cardioplegia (Blebbistatin) is added in the high and low concentrations of dyeing preparations. For details, see the above formula. If there is still a violent heart beat when recording on the machine (generally not), the cardioplegia can be added again.

[0034] 6. In addition, compared with animals such as rats and mice, the zebrafish used in the present invention has electrocardiographic characteristics that are more similar to human electrophysiological indicators, which is more conducive to the study of human-related diseases.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The method of the present invention can meet the relevant indicators required in electrophysiology literature to describe calcium signals, such as Active time, CaD, CaD despersion, Rise time and other indicators, and can especially reflect the changes in calcium signals in cells. In addition, since the heart of young zebrafish is small, very few calcium-sensitive dyes are required, and there is no need to wait until it reaches adulthood to conduct experiments, so the present invention also has the advantages of low cost and short experimental cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Comparison of cardiac thermal images of the WT group and the VPH modeling group on different days in Example 1 (unit: ms);

[0038] Figure 2 For Example 1 Figure 1 Representative calcium signal waveforms of the WT group;

[0039] Figure 3 For Example 1 Figure 1 Representative calcium signal waveforms of the VPH modeling group;

[0040] Figure 4 This is a picture of the separation device used in Comparative Example 1 (1 ml syringe);

[0041] Figure 5 The fluorescent morphological images of the two damaged hearts separated in Comparative Example 1;

[0042] Figure 6 The calcium signal waveforms of the two damaged hearts separated in Comparative Example 1 are shown;

[0043] Figure 7 is the fluorescent morphology of the heart separated in Comparative Example 2;

[0044] Figure 8 This is the calcium signal waveform of the isolated heart in Comparative Example 2;

[0045] Fig. 9 is the fluorescent morphology of the heart separated in Comparative Example 3;

[0046] Fig.10 2. The calcium signal waveform and heat map of the isolated heart in Comparative Example 3. DETAILED DESCRIPTION

[0047] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the methods, steps or conditions of the present invention are within the scope of the present invention. The products, equipment, instruments, etc. used in the following examples are commercially available unless otherwise specified, and the methods used are consistent with conventional methods unless otherwise specified.

[0048] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0049] Example 1

[0050] This embodiment provides a zebrafish heart calcium signal detection method based on an isolated zebrafish heart electrophysiological experimental method, and the specific steps are as follows:

[0051] (1) Prepare Tyrode's solution that can maintain zebrafish cardiac activity: it consists of 136 mM NaCl, 5.4 mM KCl, 1 mM MgCl2·6H2O, 10 mM HEPES (Sangon Biotech, CAS: [75277-39-3]), 5 mM D-glucose (Sangon Biotech, CAS# [50-99-7]), 1.8 mM CaCl2·2H2O, and 0.3 mM Na2HPO4·2H2O. After preparation, adjust the pH value to 7.4 with NaOH, then filter, oxygenate for 20 min, and then place in a 28°C water bath for use.

[0052] (2) First, prepare the stock solutions of Rhod2 (Abcam ab142780) (calcium-sensitive dye) and Blebbistatin (Macklin B873251-5mg) (cardioplegic agent) with DMSO, and F127 (invitrogen P3000MP) (punched) can be used directly. Then use these reagents to prepare the dye solution and the cardioplegic solution, where:

[0053] Low concentration calcium-sensitive infection solution: 1 system = Rhod2 (7 μM) + Blebbistatin (10 μM) + Tyrode's solution to make up; when using, add 2 ul F127 to every 1 ml of the system.

[0054] High concentration calcium-sensitive infection solution: 1 system = Rhod2 (14 μM) + Blebbistatin (10 μM) + Tyrode's solution to make up; when using, add 2 ul F127 to every 1 ml of the system.

[0055] Cardioplegic solution: 1 system = Blebbistatin (10 μM) + Tyrode's solution to make up.

[0056] (3) Staining method: We can use 3dpf zebrafish for the experiment at the earliest. First, the zebrafish is subjected to hypothermic shock treatment, and then the complete heart is separated in a small glass dish (using an acupuncture needle. Specifically, the needle body is reinforced with tape to expose a little of the needle tip. The specific separation steps are: 1. Cut off the inflow and outflow tracts of the heart to separate the pericardial cavity; 2. Separate the residual tissue fragments in the pericardial cavity to obtain a complete heart). Then use a pipette to suck away the Tyrode solution and excess tissue fragments, and use a pipette to absorb high-concentration calcium-sensitive infection solution to immerse the heart in light-proof staining, which can make the dye penetration more uniform. After that, the dye is sucked away, and low-concentration calcium-sensitive infection solution is absorbed again to immerse the heart on a shaker in light-proof staining for 10 minutes. After the staining is completed, the dye is sucked away, and finally the cardioplegia solution is added to immerse the heart and the data can be recorded on the computer (MappingLab electrophysiology instrument) (The entire staining process does not involve the transfer of the heart, and the small dish can be directly taken to the computer to record the data).

[0057] Comparison of the hearts of the WT group and the VPH model group detected on different days using the method in this embodiment is shown in the figure below. Figure 1 The representative calcium signal waveforms of the WT group and the VPH model group are shown in Figure 2 and Figure 3 shown.

[0058] first, Figure 1 It can be verified that the method in the present invention can indeed be implemented, and the heat map is also complete. Figure 1 to Figure 3 The specific differences in the heat maps between the WT group and the VPH group can be seen in Figure 3 The calcium transients have a normal duration, stable periodicity, and smooth waveforms. Figure 4 It shows that the time course and rhythmicity of the cycle of calcium transients are destroyed and the calcium transient waves are relatively disordered. Therefore, these figures well demonstrate the maturity of the technology of the present invention and the interpretability of the data. That is, the method of the present invention can not only meet the specific requirements of the relevant indicators that can describe calcium signals required in the electrophysiology-related literature, such as Active time, CaD, CaD despersion, Rise time and other data, but also because the heart of young zebrafish is small, the required calcium sensitive dye is also very small, and there is no need to wait until it grows up to carry out the experiment, so the present invention also has the advantages of low cost and short experimental cycle.

[0059] During the implementation of this embodiment, the present invention also conducted experiments on several other Tyrode's solution formulas with different specific dosages of raw material components from the present invention. The results showed that the above improved formula in the present invention can ensure a longer cardiac activity time.

[0060] Comparative Example 1

[0061] Since the heart of a normal zebrafish fry is very fragile and difficult to separate, the heart of a zebrafish fry with heart failure is even more fragile and more difficult to separate. Therefore, in order to fully highlight the difference between the heart separation method of the present invention and the conventional method, this comparative example uses the heart of a normal zebrafish fry as the separation object. In this comparative example, a 1 ml syringe is used as the separation needle. Figure 4 Other separation methods are consistent with those in Example 1. Figure 5 and Figure 6 The fluorescent morphological images of two damaged hearts separated by the comparative example method and their corresponding calcium signal waveform images are shown.

[0062] The results showed that the 1ml syringe as a separation needle could not achieve the required precision, and the separated hearts were incomplete, with defects in the atria and ventricles. In addition, because the heart was damaged, it was easy to inactivate, resulting in the instrument failing to record or recording bad data, and failing to generate a heat map of data such as Active Time.

[0063] Comparative Example 2

[0064] The staining method in this comparative example is to use a single low-concentration calcium-sensitive infection solution for staining, and other methods are the same as those in Example 1.

[0065] Single low-concentration calcium-sensitive infection solution staining method: the concentrations of other components remain unchanged, only the concentration of Rhod2 is changed, staining for 10-15 minutes, and other steps remain unchanged. That is, 1 system = Rhod2 (7μM) + Blebbistatin (10μM) + Tyrode's solution to make up; and when it is used specifically, 2μl F127 needs to be added to each 1mL system.

[0066] Figure 7 The obtained cardiac fluorescence morphology is shown in Figure 2. Figure 8 This is the calcium signal waveform.

[0067] Compared with the damaged heart in Comparative Example 3, Figure 7 The complete heart morphology can be clearly shown, which contains two spherical structures, one atrium and one ventricle. At the same time, because the dye concentration is too low in this case, the heart is not stained with fluorescence, so Figure 7 It shows that the heart fluorescence intensity is already very dim at this time. And because of this, Figure 8 As shown in the waveform diagram, the instrument cannot record calcium signal data, let alone generate a heat map. Therefore, there is no need to explore experimental conditions at lower concentrations.

[0068] In addition, regarding the staining time: although the vitality can be maintained and prolonged to a certain extent in Tyrode's solution, the vitality of the isolated heart is limited, so the basic staining time is not adjusted much. The following staining methods are all basically for comparison within 10-15 minutes, plus separation, operation intervals, and machine operation also take time.

[0069] Comparative Example 3

[0070] The dyeing method in this comparative example is to use a single high-concentration calcium-sensitive dye for dyeing, and other methods are the same as those in Example 1.

[0071] Single high-concentration calcium-sensitive infection solution staining method: the concentrations of other components remain unchanged, only the concentration of Rhod2 is changed, staining for 10-15 minutes, and other steps remain unchanged. That is, 1 system = Rhod2 (15-20μM) + Blebbistatin (10μM) + Tyrode's solution to make up; and when it is used specifically, 2μl F127 needs to be added to each 1mL system.

[0072] Fig. 9 The obtained cardiac fluorescence morphology is shown in Figure 2. Fig.10 The calcium signal waveform and heat map are shown.

[0073] Fig. 9 and Fig.10 It is shown that fluorescence overexposure will occur at 15-20μM. In this case, it is possible to record calcium signals, but it will cause the heat map of the data to be incomplete and the desired complete data results will not be obtained. Therefore, there is no need to explore higher concentrations.

[0074] Comparative Example 4

[0075] This comparative example is the in vivo electrophysiological experimental method introduced in the background technology, and the main steps are as follows:

[0076] Preparation stage:

[0077] 1. Select healthy juvenile zebrafish and ensure that their age and size are suitable for the experiment.

[0078] 2. Use transparent skin technology to facilitate observation and recording of neuronal activity.

[0079] Fixation and sectioning:

[0080] 1. The zebrafish should be properly anesthetized to reduce its discomfort.

[0081] 2. Use a microscope to image the zebrafish brain to ensure that the morphology and connections of neurons can be clearly seen.

[0082] Electrophysiological recordings:

[0083] 1. Electrophysiological recordings from zebrafish hearts and neurons using all-optical electrophysiology tools. These tools can capture electrical signals from heartbeats and neural activity and assess underlying electrical excitability and connectivity.

[0084] 2. Observe the entire brain of zebrafish through a high-magnification objective lens (10-20 times), and perform in vivo electrophysiological or optical imaging recording of neuronal activity on a whole-brain scale.

[0085] This method has the disadvantages of high cost and long experimental cycle.

[0086] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Tyrode's solution capable of maintaining the activity of zebrafish isolated heart, characterized in that: The Tyrode's solution consists of 130-150 mM NaCl, 4-6 mM KCl, 0.5-1.5 mM MgCl2·6H2O, 5-10 mM HEPES, 5-10 mM D-glucose, 1.5-2.0 mM CaCl2·2H2O and 0-0.5 mM Na2HPO4·2H2O.

2. A zebrafish isolated heart dye solution based on the Tyrode's solution of claim 1, characterized in that: It is a combination of a low-concentration calcium-sensitive infection solution and a high-concentration calcium-sensitive infection solution.

3. The zebrafish isolated heart dye solution according to claim 2, characterized in that The low-concentration calcium-sensitive infection solution of each system includes 6-8 μM Rhod2 and 9-11 μM Blebbistatin, and the high-concentration calcium-sensitive infection solution of each system includes 13-14 μM Rhod2 and 9-11 μM Blebbistatin, and the rest is made up by the Tyrode's solution described in claim 1; and when the low-concentration calcium-sensitive infection solution and the high-concentration calcium-sensitive infection solution are used, 1-3 μl F127 needs to be added to each 1 ml system.

4. A zebrafish isolated cardioplegia solution based on the Tyrode's solution of claim 1, characterized in that: The zebrafish isolated heart arrest solution in each system includes 9-11 μM Blebbistatin, and the remainder is made up by the Tyrode's solution described in claim 1.

5. A zebrafish cardiac electrophysiological detection method, characterized in that: The method comprises using the zebrafish isolated heart dye solution described in any one of claims 2 to 3 and / or the zebrafish isolated heart arrest solution described in claim 4.

6. The zebrafish cardiac electrophysiological detection method according to claim 5, characterized in that: The method comprises the following steps: (1) Select juvenile zebrafish and isolate the intact heart by hypothermic shock; (2) using the high concentration calcium-sensitive infection solution to immerse the heart for staining; (3) removing the high-concentration calcium-sensitive infection solution and using the low-concentration calcium-sensitive infection solution to immerse the heart for staining; (4) removing the low-concentration calcium-sensitive infection solution and submerging the heart with the cardioplegia solution; (5) Computer records.

7. The zebrafish cardiac electrophysiological detection method according to claim 6, characterized in that: In the step (1), the earliest zebrafish that can be used is 3 dpf, and the heart is separated using an acupuncture needle. The acupuncture needle is processed by reinforcing the needle body with tape to expose a little of the needle tip.

8. The zebrafish cardiac electrophysiological detection method according to claim 7, characterized in that: The staining method in step (2) is to slowly shake on a shaker for 1 to 3 minutes, and the staining method in step (3) is to slowly shake on a shaker for 8 to 12 minutes.

9. A method for isolating zebrafish heart, characterized in that: The method is the zebrafish heart isolation method used in any one of the zebrafish heart electrophysiological detection methods of claims 6 to 8.

10. Use of the Tyrode's solution described in claim 1, the zebrafish isolated heart dye solution described in any one of claims 2 to 3, the zebrafish isolated heart arrest solution described in claim 4, the zebrafish heart electrophysiological detection method described in any one of claims 5 to 8, and / or the zebrafish heart isolation method described in claim 9 in zebrafish heart-related experimental research, disease research, and / or subsequent product transformation.

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