A method and tool for regulating apoptosis of cardiomyocytes

Abstract

The present invention provides a method and tool for regulating cardiomyocyte apoptosis, which relates to the field of cell technology. The method can accurately and effectively regulate the process of cardiomyocyte apoptosis by transfecting a miRNA precursor that inhibits cell apoptosis into cardiomyocytes, and adding nutrients that produce ATP to the culture medium at different apoptosis stages during the process of cardiomyocyte apoptosis to slow down, inhibit or reverse apoptosis.

Description

Technical Field

[0001] The present invention relates to the field of cell technology, and in particular to a method for regulating apoptosis of cardiomyocytes and a tool cardiomyocyte. Background Art

[0002] In the field of cardiovascular disease research and treatment, cardiomyocyte apoptosis is a core link in the development of many heart diseases such as myocardial infarction and heart failure. When cardiomyocyte apoptosis occurs, the normal tissue structure and function of the heart are severely damaged, resulting in a decrease in the heart's ability to pump blood, which in turn causes a series of serious cardiovascular symptoms, greatly threatening the patient's life, health and quality of life. Therefore, effective regulation of cardiomyocyte apoptosis has become one of the key tasks in cardiovascular disease research, and in-depth exploration of the mechanism of cardiomyocyte apoptosis is the cornerstone of developing targeted treatment strategies.

[0003] At the basic research level, the elucidation of the mechanism of cell apoptosis depends on accurate and reliable cell models. Traditional cardiomyocyte models have exposed many limitations when simulating disease states or studying the regulatory mechanisms of cell apoptosis. For example, some in vitro cultured cardiomyocyte models are difficult to accurately reproduce the complex physiological environment in vivo, and factors such as cell-cell interactions, the influence of the extracellular matrix, and neurohumoral regulation are difficult to be fully reflected in traditional models. This makes the research results obtained based on these models likely to deviate from the actual situation in vivo, which to some extent hinders the comprehensive and in-depth understanding of the mechanism of cardiomyocyte apoptosis.

[0004] In terms of drug development, the limitations of traditional cardiomyocyte models are also significant. At present, the development of drugs for cardiovascular diseases urgently needs efficient and accurate screening models. However, the existing cardiomyocyte models are not accurate enough in simulating the characteristics of cell apoptosis in disease states and cannot accurately reflect the intervention effects of drugs on cardiomyocyte apoptosis in vivo. This has led to a large number of potential drugs being misjudged in the early cell model screening stage during the drug development process, which not only wastes a lot of manpower, material resources and time resources, but also makes many potential anti-apoptotic drugs difficult to discover and develop.

[0005] With the continuous deepening of life science research, cell engineering technology and molecular biology methods have made great progress. People have gradually realized that it is of great significance to develop a new type of cardiomyocyte model, namely tool cardiomyocytes, which can precisely regulate the apoptosis process through specific methods. This type of tool cardiomyocyte can more accurately simulate the apoptosis process of cardiomyocytes in different disease states in vitro, providing a more ideal platform for in-depth exploration of the molecular mechanism of cardiomyocyte apoptosis. By performing various experimental operations on tool cardiomyocytes, such as gene editing, signal pathway intervention, etc., the effects of different factors on cardiomyocyte apoptosis can be systematically studied, thereby revealing the key regulatory nodes in the process of cardiomyocyte apoptosis.

[0006] In terms of anti-apoptotic drug screening, tool cardiomyocytes can be used as an efficient screening tool. Because their apoptosis process can be precisely regulated, the drugs to be screened can be applied to tool cardiomyocytes, and the inhibitory effect of drugs on cardiomyocyte apoptosis can be quickly and accurately evaluated by observing changes in cell apoptosis-related indicators, such as the expression level of apoptotic proteins, cell morphological changes, mitochondrial functional status, etc. This helps to improve the efficiency and accuracy of drug screening and accelerate the development of anti-apoptotic drugs.

[0007] In summary, the development of a tool cardiomyocyte that regulates the apoptosis process based on a specific method can effectively make up for the shortcomings of traditional cardiomyocyte models in studying the mechanism of cardiomyocyte apoptosis, drug development, and cell therapy, fill the current technical gap in the precise regulation of cardiomyocyte apoptosis and the construction of related cell models, and provide strong support and promotion for the further development of cardiovascular disease research. Summary of the invention

[0008] One of the purposes of the present invention is to provide a method for regulating cardiomyocyte apoptosis, wherein the method comprises first transfecting a miRNA precursor that inhibits cell apoptosis into cardiomyocytes, and then, during the process of cardiomyocyte apoptosis, adding nutrients that produce ATP to the culture medium to regulate the process of cardiomyocyte apoptosis.

[0009] Preferably, the miRNA precursor that inhibits cell apoptosis includes one or more of miRNA-671 precursor, miR-133 precursor, miR-21 precursor or miR-499 precursor.

[0010] Preferably, Dicer enzyme inhibition is performed before transfection of the miRNA precursor that inhibits cell apoptosis, and the inhibition is released after the transfection is completed.

[0011] Preferably, the ATP-producing nutrients include one or more of glucose, glycogen, fructose-1,6-bisphosphate, palmitic acid, oleic acid, β-hydroxybutyric acid, glutamic acid, aspartic acid, sodium pyruvate or sodium succinate.

[0012] Preferably, the nutrients that produce ATP also include vitamin C, vitamin E, N-acetylcysteine, potassium chloride, magnesium chloride, calcium chloride, sodium gluconate, arginine, lysine, glycine and brain-derived neurotrophic factor.

[0013] Preferably, the nutrients that produce ATP include nutrients for the early apoptosis stage, nutrients for the middle apoptosis stage, and nutrients for the late apoptosis stage.

[0014] Preferably, the amount of nutrients added in the early apoptosis stage is 5%-15% (v / v) of the culture medium, the amount of nutrients added in the middle apoptosis stage is 3%-10% (v / v) of the culture medium, and the amount of nutrients added in the late apoptosis stage is 1%-5% (v / v) of the culture medium.

[0015] Preferably, the ingredients of the early apoptosis stage nutrients include: Energy substrates: sodium pyruvate 5 mM, sodium succinate 3 mM, glutamine 4 mM, glucose 10 mM, and fructose-1,6-bisphosphate 1 mM; Antioxidants: vitamin C 300 μM, vitamin E 150 μM, and N-acetylcysteine ​​400 μM; Electrolytes and osmotic pressure regulators: potassium chloride 5mM, magnesium chloride 1.5mM, calcium chloride 2.5mM, and sodium gluconate 4mM; Amino acids: arginine 0.8 mM, lysine 0.6 mM, glycine 0.5 mM, glutamate 0.5, and aspartic acid 0.4 mM; Neurotrophic factors: brain-derived neurotrophic factor 20 ng / ml, and Buffer: HEPES buffer 30 mM.

[0016] Preferably, the ingredients of the mid-apoptotic stage nutrients include: Energy substrates: sodium pyruvate 3.5 mM, sodium succinate 2.5 mM, glutamine 3 mM, glucose 8 mM, and fructose-1,6-diphosphate 0.8 mM; Antioxidants: vitamin C 200 μM, vitamin E 100 μM, and N-acetylcysteine ​​300 μM; Electrolytes and osmotic pressure regulators: potassium chloride 4.5 mM, magnesium chloride 1.3 mM, calcium chloride 2.2 mM, and sodium gluconate 5 mM; Amino acids: arginine 0.65 mM, lysine 0.5 mM, glycine 0.4 mM, glutamate 0.4, and aspartic acid 0.3 mM; Neurotrophic factors: brain-derived neurotrophic factor 25 ng / ml, and Buffer: HEPES buffer 25 mM.

[0017] Preferably, the ingredients of the late apoptosis stage nutrients include: Energy substrates: sodium pyruvate 2 mM, sodium succinate 2 mM, glutamine 2 mM, glucose 5 mM, and fructose-1,6-diphosphate 0.5 mM; Antioxidants: vitamin C 100 μM, vitamin E 50 μM, and N-acetylcysteine ​​200 μM; Electrolytes and osmotic pressure regulators: potassium chloride 4mM, magnesium chloride 1.1mM, calcium chloride 2mM, and sodium gluconate 6mM; Amino acids: arginine 0.5 mM, lysine 0.4 mM, glycine 0.3 mM, glutamate 0.3, and aspartic acid 0.2 mM; Neurotrophic factors: Brain-derived neurotrophic factor 30 ng / ml, and Buffer: HEPES buffer 20 mM.

[0018] A second object of the present invention is to provide a tool cardiomyocyte.

[0019] Beneficial effects of the present invention: The present invention provides a method and tool cardiomyocytes that can regulate cardiomyocyte apoptosis. The method can accurately and effectively regulate the process of cardiomyocyte apoptosis. By transfecting miRNA precursors that inhibit cell apoptosis into cardiomyocytes, and adding nutrients that produce ATP to the culture medium during the process of cardiomyocyte apoptosis, effective intervention and regulation can be performed both in the signal transduction link at the beginning of apoptosis and in the key molecular change stage during the execution of apoptosis. Therefore, researchers can inhibit cell apoptosis at any time according to actual needs. For cardiomyocytes that are already in the process of apoptosis, it can activate the self-repair and regeneration mechanism in the cells, promote the reversal of the physiological state of the cells to normal, and restore their survival ability and functional activity. Tool cardiomyocytes that are transfected with miRNA precursors that inhibit cell apoptosis can slow down, inhibit or reverse apoptosis by adding nutrients that produce ATP at different stages of apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the flow cytometry graph of the normal group cells after 15 hours of culture; Figure 2 This is the flow cytometry graph of the model group cells after 2 hours of culture; Figure 3 This is the flow cytometry graph of the model group cells after 6 hours of culture; Figure 4 This is the flow cytometry graph of the model group cells after 10 hours of culture; Figure 5 This is the flow cytometry graph of the control group 1; Figure 6 The flow cytometry graphs of the two control groups; Figure 7 The flow cytometry graphs of the three control groups; Figure 8 Flow cytometry of 4 groups of cells are shown as controls; Fig. 9 This is the flow cytometry graph of the cells in the experimental group 1; Fig.10 This is the flow cytometry diagram of the cells in the experimental group 2; Fig.11 This is the flow cytometry graph of the three experimental groups; Fig.12 This is the flow cytometry graph of the 4 experimental groups; Fig.13 This is the flow cytometry graph of the 5 experimental groups; Fig.14 The figure shows the flow cytometry graphs of the six experimental groups. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0022] The technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0023] Example 1. Experimental Materials H9c2 cardiomyocytes, which have good growth characteristics; GFP-tagged miRNA precursor; Lipofectamine2000 high-efficiency transfection reagent; Cell culture medium: DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution for routine cell culture and maintenance; Sugar-free EBSS medium; AnnexinV-FITC / PI apoptosis detection kit.

[0024] 2. Cell Processing and Grouping Cardiomyocyte culture and preconditioning The cardiomyocytes were inoculated into cell culture dishes and cultured in a 37°C, 5% CO2 incubator. When the cells grew to the logarithmic growth phase and the density reached 60%-70%, the medium was replaced with sugar-free EBSS medium and cultured in a hypoxic incubator (1%-2% O2). Samples were taken at 2, 4, 6, 8, 10, 12, 16, 20, and 24 hours of culture.

[0025] Take a portion of the cell samples and observe the cell morphology under an optical microscope. Record the time when the cells begin to become smaller and rounder, and the connection between cells becomes loose; the time when the nuclear chromatin is observed to condense around the nuclear membrane; and the time when the cells break into apoptotic bodies.

[0026] For another part of the cell samples at each time point, the staining operation was performed according to the instructions of the AnnexinV-FITC / PI apoptosis detection kit. Then the normal cells (AnnexinV-FITC - / PI- ), early apoptotic cells (AnnexinV-FITC + / PI - ) and late apoptotic cells (AnnexinV-FITCI + / PI + ) ratio changes. + / PI - When the cell ratio begins to increase significantly and the early apoptotic morphological characteristics appear under the optical microscope, the early apoptosis onset time range can be determined; when AnnexinV-FITC + / PI + When the cell ratio increases rapidly and the mid-apoptotic morphology is observed under an optical microscope, the mid-apoptotic time range can be roughly determined; when most of the cells show AnnexinV-FITC + / PI + When a large number of apoptotic bodies appear, it is determined to be in the late apoptosis time range.

[0027] Combined with the results of optical microscopy observations, it was determined that the time range for early apoptosis was 2 to 4 hours, the time range for mid-apoptosis was 4 to 8 hours, and the time range for late apoptosis was 8 to 15 hours.

[0028] The cardiomyocytes were divided into normal group, model group, control group 1, control group 2, control group 3, control group 4, experimental group 1, experimental group 2, experimental group 3, experimental group 4, experimental group 5 and experimental group 6. The treatment method of each group was as follows: Normal group: The cardiomyocytes were cultured in DMEM medium containing 10% serum at 37°C and 5% CO2 for 15 hours without glucose and oxygen deprivation treatment.

[0029] Model group: The cardiomyocytes were cultured in a sugar-free EBSS medium in a hypoxic incubator (1%-2% O2) for 15 hours to induce a cardiomyocyte apoptosis model. Samples were taken at 2, 6, and 10 hours for cell apoptosis detection.

[0030] Control group 1: The cardiomyocytes were cultured in DMEM medium containing 0.2 μmol / L Dicer enzyme inhibitor for 4-6 hours, the medium containing Dicer enzyme inhibitor was carefully aspirated, and then replaced with preheated fresh medium without inhibitors to transfect the cardiomyocytes with miRNA-671 precursor. According to the instructions of the transfection reagent, the miRNA-671 precursor was gently mixed with Lipofectamine2000 in serum-free medium and incubated at room temperature for 15-20 minutes to form a transfection complex. The transfection complex was added dropwise to the cardiomyocyte culture well, gently shaken, and placed in a 37°C, 5% CO2 incubator for continued culture. After 24-48 hours of transfection, the expression of the fluorescently labeled miRNA-671 precursor in the cells was observed by fluorescence microscopy to evaluate the transfection efficiency. Multiple fields of view were selected to count the number of fluorescent positive cells and the total number of cells, and the transfection efficiency was calculated to ensure that the transfection efficiency reached more than 70%. Then, glucose and hypoxia treatment was performed in the same way as the model group, and cell apoptosis was detected 10 hours after treatment.

[0031] Control group 2: After the cardiomyocytes were treated with glucose and oxygen deprivation for 2 hours, 10% of the volume of culture medium was added with nutrients in the early apoptosis stage, and the cells were cultured at 37°C and 5% CO2 for 15 hours to detect cell apoptosis.

[0032] Control group 3: After the cardiomyocytes were treated with glucose and oxygen deprivation for 6 hours, 7% of the volume of culture medium of mid-apoptosis stage nutrients were added, and the cells were cultured at 37°C and 5% CO2 for 15 hours to detect cell apoptosis.

[0033] Control group 4: After the cardiomyocytes were treated with glucose and oxygen deprivation for 10 hours, 3% of the volume of culture medium of late apoptosis stage nutrients were added, and the cells were cultured at 37°C and 5% CO2 for 15 hours to detect cell apoptosis.

[0034] Experimental group 1: cardiomyocytes were cultured in DMEM medium containing 0.2 μmol / L Dicer enzyme inhibitor for 4-6 hours, the medium containing Dicer enzyme inhibitor was carefully aspirated, and then replaced with preheated fresh medium without inhibitor, and the cardiomyocytes were transfected with miRNA-671 precursor. The transfection method was the same as that of control group 1. After 2 hours of glucose and oxygen deprivation treatment, 5% (v / v) early apoptosis stage nutrients were added to the culture medium, and the cells were cultured at 37°C and 5% CO2 for 15 hours to detect cell apoptosis.

[0035] Experimental group 2: cardiomyocytes were cultured in DMEM medium containing 0.2 μmol / L Dicer enzyme inhibitor for 4-6 hours, the medium containing Dicer enzyme inhibitor was carefully aspirated, and then replaced with preheated fresh medium without inhibitor, and the cardiomyocytes were transfected with miRNA-671 precursor. The transfection method was the same as that of control group 1. After 2 hours of glucose and oxygen deprivation treatment, 15% (v / v) early apoptosis stage nutrients were added to the culture medium, and the cells were cultured at 37°C and 5% CO2 for 15 hours to detect cell apoptosis.

[0036] Experimental group 3: cardiomyocytes were cultured in DMEM medium containing 0.2 μmol / L Dicer enzyme inhibitor for 4-6 hours, the medium containing Dicer enzyme inhibitor was carefully aspirated, and then replaced with preheated fresh medium without inhibitor, and the cardiomyocytes were transfected with miRNA-671 precursor. The transfection method was the same as that of control group 1. After 6 hours of glucose and hypoxia treatment, 3% (v / v) mid-apoptosis stage nutrients were added to the culture medium, and the cells were cultured at 37°C and 5% CO2 for 15 hours to detect cell apoptosis.

[0037] Experimental group 4: cardiomyocytes were cultured in DMEM medium containing 0.2 μmol / L Dicer enzyme inhibitor for 4-6 hours, the medium containing Dicer enzyme inhibitor was carefully aspirated, and then replaced with preheated fresh medium without inhibitor, and the cardiomyocytes were transfected with miRNA-671 precursor. The transfection method was the same as that of control group 1. After 6 hours of glucose and oxygen deprivation treatment, 10% (v / v) mid-apoptosis stage nutrients were added to the culture medium, and the cells were cultured at 37°C and 5% CO2 for 15 hours to detect cell apoptosis.

[0038] Experimental group 5: The cardiomyocytes were cultured in DMEM medium containing 0.2 μmol / L Dicer enzyme inhibitor for 4-6 hours, the medium containing Dicer enzyme inhibitor was carefully aspirated, and then replaced with preheated fresh medium without inhibitor, and the cardiomyocytes were transfected with miRNA-671 precursor. The transfection method was the same as that of control group 1. After 10 hours of glucose and oxygen deprivation treatment, 1% (v / v) late apoptosis stage nutrients were added to the culture medium, and the cells were cultured at 37°C and 5% CO2 for 15 hours to detect cell apoptosis.

[0039] Experimental group 6: cardiomyocytes were cultured in DMEM medium containing 0.2 μmol / L Dicer enzyme inhibitor for 4-6 hours, the medium containing Dicer enzyme inhibitor was carefully aspirated, and then replaced with preheated fresh medium without inhibitor, and the cardiomyocytes were transfected with miRNA-671 precursor. The transfection method was the same as that of control group 1. After 10 hours of glucose and oxygen deprivation treatment, 5% (v / v) late apoptosis stage nutrients were added to the culture medium, and the cells were cultured at 37°C and 5% CO2 for 15 hours to detect cell apoptosis.

[0040] 2. Cell Apoptosis Detection AnnexinV-FITC / PI double staining method: The cardiomyocytes from different treatment groups were collected, washed twice with pre-cooled PBS, and then resuspended in Binding Buffer to adjust the cell concentration to about 1×10 6 / ml. Take 100μl of cell suspension, add 5μl AnnexinV-FITC and 5μl PI staining solution, mix gently, and incubate at room temperature in the dark for 15 minutes. Add 400μl Binding Buffer, mix well and immediately detect cell apoptosis using flow cytometry.

[0041] The horizontal axis represents the fluorescence intensity of AnnexinV-FITC, and the vertical axis represents the fluorescence intensity of PI. In the scatter plot, four quadrants appear: Lower left quadrant (AnnexinV-FITC - / PI - ): represents normal living cells, the cell membrane of these cells is intact, PS is not everted, and is not stained by PI.

[0042] Lower right quadrant (AnnexinV-FITC + / PI - ): represents early apoptotic cells. The PS of these cells is externalized and can bind to AnnexinV-FITC, but the cell membrane is still intact and PI cannot enter the cells.

[0043] Upper right quadrant (AnnexinV-FITCI + / PI + ): represents late apoptotic cells or necrotic cells, the PS of the cells is everted, and the cell membrane integrity is lost, and PI can enter the cells.

[0044] Upper left quadrant (AnnexinV-FITCI - / PI + ): This is usually caused by atypical situations such as cell debris or damaged cells. Cells in this quadrant are generally not considered when calculating the apoptosis rate.

[0045] Apoptosis rate (%) = (number of cells in the lower right quadrant + number of cells in the upper right quadrant) / (number of cells in the lower left quadrant + number of cells in the lower right quadrant + number of cells in the upper right quadrant) × 100% The cell apoptosis rate was determined by analyzing the ratio of cells in different quadrants. Figure 1-14 The cell apoptosis in each group is shown in Table 1.

[0046] Table 1 Cell apoptosis in each group

Claims

1. A method for regulating cardiomyocyte apoptosis, characterized in that: The method comprises first transfecting a miRNA precursor that inhibits cell apoptosis into cardiomyocytes, and then adding nutrients that produce ATP to the culture medium during the process of cardiomyocyte apoptosis to regulate the process of cardiomyocyte apoptosis.

2. The method for regulating cardiomyocyte apoptosis according to claim 1, characterized in that: The miRNA precursors that inhibit cell apoptosis include one or more of miRNA-671 precursor, miR-133 precursor, miR-21 precursor or miR-499 precursor.

3. The method for regulating cardiomyocyte apoptosis according to claim 1, characterized in that: The ATP-producing nutrients include one or more of glucose, fructose-1,6-diphosphate, palmitic acid, oleic acid, β-hydroxybutyric acid, glutamic acid, aspartic acid, sodium pyruvate or sodium succinate.

4. The method for regulating cardiomyocyte apoptosis according to claim 1, characterized in that: The ATP-producing nutrients also include vitamin C, vitamin E, N-acetylcysteine, potassium chloride, magnesium chloride, calcium chloride, sodium gluconate, arginine, lysine, glycine and brain-derived neurotrophic factor.

5. The method for regulating cardiomyocyte apoptosis according to claim 1, characterized in that: The nutrients that produce ATP include nutrients in the early apoptosis stage, nutrients in the middle apoptosis stage, and nutrients in the late apoptosis stage.

6. The method for regulating cardiomyocyte apoptosis according to claim 5, characterized in that: The amount of nutrients added in the early apoptosis stage is 5%-15% (v / v) of the culture medium, the amount of nutrients added in the middle apoptosis stage is 3%-10% (v / v) of the culture medium, and the amount of nutrients added in the late apoptosis stage is 1%-5% (v / v) of the culture medium.

7. The method for regulating cardiomyocyte apoptosis according to claim 5, characterized in that: The components of the early apoptosis stage nutrients include: Energy substrates: sodium pyruvate 5 mM, sodium succinate 3 mM, glutamine 4 mM, glucose 10 mM, and fructose-1,6-bisphosphate 1 mM; Antioxidants: vitamin C 300 μM, vitamin E 150 μM, and N-acetylcysteine ​​400 μM; Electrolytes and osmotic pressure regulators: potassium chloride 5mM, magnesium chloride 1.5mM, calcium chloride 2.5mM, and sodium gluconate 4mM; Amino acids: arginine 0.8 mM, lysine 0.6 mM, glycine 0.5 mM, glutamate 0.5, and aspartic acid 0.4 mM; Neurotrophic factors: brain-derived neurotrophic factor 20 ng / ml, and Buffer: HEPES buffer 30 mM.

8. The method for regulating cardiomyocyte apoptosis according to claim 5, characterized in that: The components of the mid-apoptosis stage nutrients include: Energy substrates: sodium pyruvate 3.5 mM, sodium succinate 2.5 mM, glutamine 3 mM, glucose 8 mM, and fructose-1,6-diphosphate 0.8 mM; Antioxidants: vitamin C 200 μM, vitamin E 100 μM, and N-acetylcysteine ​​300 μM; Electrolytes and osmotic pressure regulators: potassium chloride 4.5 mM, magnesium chloride 1.3 mM, calcium chloride 2.2 mM, and sodium gluconate 5 mM; Amino acids: arginine 0.65 mM, lysine 0.5 mM, glycine 0.4 mM, glutamate 0.4, and aspartic acid 0.3 mM; Neurotrophic factors: brain-derived neurotrophic factor 25 ng / ml, and Buffer: HEPES buffer 25 mM.

9. The method for regulating cardiomyocyte apoptosis according to claim 5, characterized in that: The components of the late apoptosis stage nutrients include: Energy substrates: sodium pyruvate 2 mM, sodium succinate 2 mM, glutamine 2 mM, glucose 5 mM, and fructose-1,6-diphosphate 0.5 mM; Antioxidants: vitamin C 100 μM, vitamin E 50 μM, and N-acetylcysteine ​​200 μM; Electrolytes and osmotic pressure regulators: potassium chloride 4mM, magnesium chloride 1.1mM, calcium chloride 2mM, and sodium gluconate 6mM; Amino acids: arginine 0.5 mM, lysine 0.4 mM, glycine 0.3 mM, glutamate 0.3, and aspartic acid 0.2 mM; Neurotrophic factors: Brain-derived neurotrophic factor 30 ng / ml, and Buffer: HEPES buffer 20 mM.

10. A tool cardiomyocyte prepared by the method for regulating cardiomyocyte apoptosis according to any one of claims 1 to 9.

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