Dynamic monitoring method for ATP (adenosine triphosphate) level in myocardial cell, recombinant myocardial cell and application

Abstract

The invention relates to the technical field of biomedicine, and particularly discloses a dynamic monitoring method for ATP (adenosine triphosphate) level in cardiac muscle cells, recombinant cardiac muscle cells and application. The preparation method comprises the following steps: cloning an iATPSnFRs gene as shown in SEQ ID NO.3 into multiple cloning sites of a pLVX-AcGFP1-N1 plasmid, so as to construct an iATPSnFR1 recombinant plasmid; then transfecting myocardial cells with the iATPSnFR1 recombinant plasmid to obtain recombinant myocardial cells; and monitoring the ATP level change in the iATPSnFR1 recombinant myocardial cells in real time by using a high content imaging system. The recombinant myocardial cell containing the iATPSnFR1 is obtained through construction, so that the iATPSnFR1 can be specifically combined with ATP in the cell and generate a fluorescence signal, real-time and dynamic monitoring of the ATP level can be realized, and the recombinant myocardial cell has the advantages of non-invasiveness, high spatial resolution and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for dynamically monitoring ATP levels in myocardial cells, recombinant myocardial cells and applications thereof. Background Art

[0002] Currently, the detection of ATP levels in cardiomyocytes primarily relies on traditional chemiluminescence and fluorescence methods, which suffer from low sensitivity and poor real-time performance. Current methods for monitoring cardiomyocyte ATP levels have significant limitations in providing real-time data. Traditional biochemical assays (such as luciferase, high-performance liquid chromatography (HPLC), and mass spectrometry) often require cell lysis to extract ATP. These methods are not only invasive but also only provide static ATP level information at a single time point, preventing continuous monitoring. Furthermore, due to factors such as ATP degradation during sample preparation and unstable extraction efficiency, measurement results are often subject to significant error. These technical limitations have severely hindered in-depth research on the dynamic changes in cardiomyocyte energy metabolism, particularly in studying real-time changes in ATP levels under various pathophysiological conditions, such as ischemia-reperfusion and metabolic stress. Therefore, a method for real-time, dynamic monitoring of ATP changes in cardiomyocytes is urgently needed. Summary of the Invention

[0003] To develop a method for real-time, dynamic monitoring of ATP changes within cardiomyocytes, the present invention provides a method for dynamic monitoring of ATP levels within cardiomyocytes, as well as recombinant cardiomyocytes and their applications. By constructing a cardiomyocyte cell line that stably expresses iATPSnFR1, the present invention enables real-time monitoring of intracellular ATP levels.

[0004] The present invention also provides a method for dynamically monitoring ATP levels in myocardial cells, wherein the iATPSnFRs gene shown in SEQ ID NO.3 is cloned into the multiple cloning site of the pLVX-AcGFP1-N1 plasmid to construct an iATPSnFR1 recombinant plasmid; The iATPSnFR1 recombinant plasmid was transformed into Escherichia coli, and positive bacterial clones containing the iATPSnFR1 recombinant plasmid were screened using antibiotic selection medium. The plasmid was extracted and transfected into cardiomyocytes. Successfully transfected cardiomyocytes were screened using puromycin to obtain iATPSnFR1 recombinant cardiomyocytes. Monitor changes in ATP levels in iATPSnFR1 recombinant cardiomyocytes.

[0005] The present invention obtains recombinant cardiomyocytes containing iATPSnFR1 by constructing and enabling iATPSnFR1 to specifically bind to ATP in the cells and generate fluorescent signals, thereby enabling real-time and dynamic monitoring of ATP levels. It also has the advantages of being non-invasive and having high spatial resolution.

[0006] Furthermore, the amplification primers for the iATPSnFRs gene include iATPSnFR1-F shown in SEQ ID NO.1 and iATPSnFR1-R shown in SEQ ID NO.2.

[0007] Furthermore, the iATPSnFRs gene was cloned into the pLVX-AcGFP1-N1 plasmid. Bam HI and Xba I site.

[0008] Furthermore, the confluence of cardiomyocytes was 70% to 80%.

[0009] Furthermore, the culture process of the cardiomyocytes with a confluence of 70% to 80% is as follows: the cardiomyocytes are inoculated into a DMEM culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin-gentamicin and cultured to a confluence of 70-80%.

[0010] The present invention also provides an iATPSnFR1 recombinant plasmid, wherein the iATPSnFRs gene shown in SEQ ID NO.3 is cloned into the pLVX-AcGFP1-N1 plasmid. Bam HI and Xba I site.

[0011] The iATPSnFR1 recombinant plasmid was constructed and obtained.

[0012] The present invention also provides a recombinant myocardial cell, which is obtained by transfecting the iATPSnFR1 recombinant plasmid into the myocardial cell.

[0013] Furthermore, the cardiomyocytes are cardiomyocyte AC16 cells.

[0014] The present invention also provides a use of the iATPSnFR1 recombinant plasmid or the recombinant myocardial cell in dynamically monitoring the ATP level in myocardial cells.

[0015] Furthermore, the iATPSnFR1 gene can specifically bind to ATP in cardiomyocytes and generate fluorescent signals, and a high-content imaging system is used to monitor changes in ATP levels in iATPSnFR1 recombinant cardiomyocytes in real time.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention obtains recombinant cardiomyocytes containing iATPSnFR1 by constructing and enabling iATPSnFR1 to specifically bind to ATP in the cells and generate fluorescent signals, thereby enabling real-time and dynamic monitoring of ATP levels. It also has the advantages of being non-invasive and having high spatial resolution.

[0017] The present invention realizes real-time and dynamic monitoring of ATP changes in myocardial cells through the development of a genetically encoded sensor iATPSnFR1. This sensor can specifically bind to ATP and generate a fluorescent signal. It can not only realize real-time and dynamic monitoring of ATP levels, but also has the advantages of being non-invasive and having high spatial resolution, providing a more accurate and reliable research tool for studying cardiac ATP metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 The cell morphology of AC16 cells expressing no pLVX-AcGFP1-N1 and overexpressing pLVX-AcGFP1-iATPSnFR1 under a normal microscope; a, b, and c are the cell morphology of AC16 cells expressing no pLVX-AcGFP1-N1 (left) and overexpressing pLVX-AcGFP1-iATPSnFR1 (right) in three fields of view (three replicates) under a 10 x 10x microscope, respectively.

[0020] Figure 2 The fluorescence signal of iATPSnFR1 overexpressing cells (AC16-iATP SnFR-OE) under a fluorescence microscope; In the figure, a represents the fluorescence signals in two different fields of view on the left and right under a 10 x 10 magnification microscope; b represents the fluorescence signals in two different fields of view at 10 × 20 magnification. c represents the fluorescence signals in two different fields of view on the left and right under a 10 x 40 magnification microscope.

[0021] Figure 3 Effects of high glucose (4.5 g / L D-glucose) and low glucose (1.0 g / L D-glucose) treatments at different time points on ATP levels in iATPSnFR1 recombinant cardiomyocytes; In the figure, a is the fluorescence intensity of cells treated with low glucose (1.0 g / L D-glucose) for 30 mins; b is the fluorescence intensity of cells treated with low glucose (1.0 g / L D-glucose) for 60 mins; c is the fluorescence intensity of cells treated with low glucose (1.0 g / L D-glucose) for 120 mins; d is the fluorescence intensity of cells treated with high glucose (4.5 g / L D-glucose) for 30 mins; e is the fluorescence intensity of cells treated with high glucose (4.5 g / L D-glucose) for 60 mins; f is the fluorescence intensity of cells treated with high glucose (4.5 g / L D-glucose) for 120 mins.

[0022] Figure 4 The effect of 0.5 μg / mL LPS on the fluorescence intensity of iATPSnFR1 recombinant cardiomyocytes; In the figure, NC represents the cell fluorescence and its intensity of the control group (treated with an equal amount of PBS solution); LPS 0.5 μg / mL represents the cell fluorescence and its intensity of the experimental group (treated with a 0.5 μg / mL dose of LPS solution); 40X represents high-content imaging under a 10 x 4 magnification microscope, and 100X represents high-content imaging under a 10 x 10 magnification microscope.

[0023] Figure 5 To quantitatively evaluate the changes in ATP levels in iATPSnFR1 recombinant cardiomyocytes treated with different concentrations of LPS; In the figure, a is a fluorescence microscope image of the changes in ATP levels in iATPSnFR1 recombinant cardiomyocytes treated with different concentrations of LPS; wherein, (1) represents the intensity of cell fluorescence under a 10 x 20 magnification microscope field of view; (2) represents the intensity of cell fluorescence under a 10 x 40 magnification microscope field of view.

[0024] b is a statistical graph showing the changes in ATP levels in iATPSnFR1 recombinant cardiomyocytes treated with different concentrations of LPS.

[0025] Figure 6 Flow cytometry was used to detect the effects of different concentrations of LPS on ATP levels in AC16 cells and iATPSnFR1 recombinant cardiomyocytes. In the figure, a represents the gate of AC16 cell clusters circled under the same FSC signal (forward scattered light) and SSC signal (side scattered light) screening conditions; b shows the fluorescence intensity and cell number of AC16 cell population corresponding to Figure a, where FITC-A + Indicates positive fluorescence signal, FITC-A - Indicates negative fluorescence signal; Figure c represents the merge of Figure b, where the red peak is the fluorescence signal peak of the negative control (Blank Control) AC16 cells, the blue peak is the fluorescence signal peak of the iATPSnFR1 recombinant cardiomyocytes treated with PBS; the orange peak is the fluorescence signal peak of the iATPSnFR1 recombinant cardiomyocytes treated with 1.5 μg / mL LPS; and the green peak is the fluorescence signal peak of the iATPSnFR1 recombinant cardiomyocytes treated with 2.5 μg / mL LPS. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0027] Example 1: A method for dynamically monitoring ATP levels in myocardial cells.

[0028] 1. Construction of recombinant plasmids and iATPSnFR1 recombinant cardiomyocytes 1. Amplification of the iATPSnFR1 gene (1) Primer design and synthesis Based on the iATPSnFRs gene sequence shown in SEQ ID NO. 3, the following primers were designed and synthesized by Shanghai Sangon Co., Ltd. The primer sequence information is shown in Table 1 below.

[0029] Table 1 Primer sequence information of iATPSnFRs gene Note: Underline indicates restriction enzyme cutting site.

[0030] (2) PCR amplification reaction The iATPSnFR1 gene fragment was amplified using polymerase chain reaction. The PCR reaction system was configured according to the Tiangen kit instructions. The specific components and amounts are shown in Table 2 below.

[0031] Table 2 PCR amplification reaction system The PCR amplification program is shown in Table 3.

[0032] The PCR amplification products were separated by agarose gel electrophoresis, and the target band was excised and purified using a gel recovery kit. The purified DNA fragment was sequenced, and the sequencing results were consistent with the sequence shown in SEQ ID NO. 3, indicating that the target gene fragment was successfully amplified.

[0033] SEQ ID NO.3:

[0034] 2. Construction of iATPSnFR1 recombinant plasmid (1) Plasmid digestion and ligation The purified iATPSnFR1 gene fragment and the pLVX-AcGFP1-N1 plasmid were double-digested with the restriction endonucleases BamHI and XbaI, respectively. The digestion reaction system and conditions were determined according to the enzyme supplier's instructions. The digestion product was purified and recovered, and then ligated using T4 DNA ligase to obtain the ligation product (iATPSnFR1 recombinant plasmid).

[0035] (2) Bacterial transformation and screening The ligation product (iATPSnFR1 recombinant plasmid) was transformed into competent Escherichia coli stbl3 cells. The transformed bacteria were plated onto LB solid medium containing 1% tryptone, 0.5% yeast extract, 1% NaCl, and 1.5% agar powder in a w / v ratio (80 μg / mL kanamycin). The cells were cultured overnight at 37°C in an incubator, and positive colonies were selected.

[0036] (3) Plasmid verification Single positive colonies growing on the plates were selected for colony PCR and plasmid DNA extraction. Positive clones were initially screened by PCR amplification and enzyme digestion analysis. The extracted plasmid DNA was sent to a sequencing company for Sanger sequencing to verify the sequence accuracy and integrity of the inserted gene.

[0037] 3. Cardiac AC16 Cell Culture and Transfection AC16 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-gentamicin to provide essential nutrients and antibiotic protection. Cells were maintained in a 37°C, 5% CO2 incubator with regular medium changes to maintain cell growth. AC16 cell growth was monitored and transfection was performed when cells reached 70% confluence. The day before transfection, cells were seeded into 6-well plates to ensure 70% confluence at the time of transfection. On the day of transfection, plasmid DNA (iATPSnFR1 recombinant plasmid) was diluted in jetPRIME® buffer. JetPRIME® reagent was then added and mixed at the recommended plasmid DNA to jetPRIME® ratio (μg mass:μL volume = 1:3). Immediately after transfection, the plate was centrifuged at 180 g for 5 minutes to facilitate DNA uptake.

[0038] Post-transfection treatment: 24 hours after transfection, replace the culture medium with fresh DMEM (containing 10% FBS) to support cell recovery and survival. Regularly observe the expression of the fluorescent protein using a fluorescence microscope and record the transfection efficiency.

[0039] The results are as follows Figure 1 As shown, transfection of iATPSnFR1 recombinant plasmid had no obvious effect on cell morphology.

[0040] 4. Screening of stable cell lines Post-transfection cell culture: 24 hours after transfection, cells were cultured at 37°C, 5% CO2. Transfected cells were plated in DMEM medium containing puromycin (1 µg / mL).

[0041] Antibiotic selection: During the culture process, unsuccessfully transfected cells are eliminated by the action of puromycin. This allows for the enrichment of cell populations carrying the target gene. During this period, the culture medium should be changed every 1-2 days to maintain the freshness of the culture medium and the cell growth environment.

[0042] Gradient screening: Gradient screening effectively selects cells with high antibiotic tolerance. This stage of culture typically lasts 7 to 10 days, during which time cell growth and morphological changes are regularly observed under a fluorescence microscope. This ensures that the resulting cell line exhibits stable expression characteristics. The resulting cell line is designated iATPSnFR1 recombinant cardiomyocytes.

[0043] The stability and expression characteristics of the monoclonal cells were confirmed by fluorescence microscopy to ensure that the obtained cell lines could continuously express the target gene. Figure 2 As described above, after screening, the positive rate of iATPSnFR1 recombinant cardiomyocytes reached more than 95%.

[0044] 2. Determination of the effects of cardiomyocyte ATP under different conditions based on iATPSnFR1 recombinant cardiomyocytes To further validate the application of iATPSnFR1 recombinant cardiomyocytes in monitoring dynamic changes in ATP levels, this example treated iATPSnFR1 recombinant cardiomyocytes with different stimuli, including lipopolysaccharide sulfate (LPS) and high-concentration (4.5 g / L) / low-concentration (1.0 g / L) glucose incubation. A high-content imaging system was then used to monitor changes in intracellular ATP levels in real time.

[0045] 1. Cell culture and treatment preparation Cell Culture: iATPSnFR1 recombinant cardiomyocytes were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-gentamicin. Cells were cultured at 37°C in a 5% CO2 incubator with regular medium changes to maintain cell growth.

[0046] Cell seeding: One day before the experiment, seed iATPSnFR1 recombinant cardiomyocytes in a high-content imaging plate or a 6-well plate. Determine the seeding density based on subsequent experimental requirements. Ensure that the cells are at 70% confluence at the time of treatment.

[0047] 2. High / low concentration glucose culture medium treatment and ATP level monitoring High / low glucose (abbreviated as high / low glucose) medium treatment: When iATPSnFR1 recombinant cardiomyocytes grow to 70% confluence, they are treated with high-glucose serum-free medium (4.5 g / L D-glucose). A low-glucose serum-free medium (1.0 g / L D-glucose) control is also provided as a parallel control.

[0048] ATP level fluorescence monitoring: Immediately after treatment with high / low glucose medium, changes in ATP levels in iATPSnFR1 recombinant cardiomyocytes were monitored in real time using a high-content imaging system with the same imaging parameters as above.

[0049] Data Acquisition and Analysis: This example focused on monitoring the rapid changes in ATP levels between 0 and 2 hours of high- and low-glucose treatment. Therefore, dynamic monitoring was performed at more frequent time points. A high-content imaging system was used to capture continuous fluorescence images of cells across multiple fields of view. These images were combined into a dynamic video, and image analysis software was used to quantitatively analyze changes in the mean fluorescence intensity of the cells.

[0050] The experimental results are as follows Figure 3 As shown, by comparing low sugar ( Figure 3 a、 Figure 3 b and Figure 3 c) with high sugar ( Figure 3 d、 Figure 3 e and Figure 3 f) Cell fluorescence intensity at 30, 60, and 120 minutes. High-glucose conditions resulted in increased ATP activity within iATPSnFR1-recombinant cardiomyocytes, and slower signal decay. This suggests that high-glucose conditions increase ATP levels within cardiomyocytes and enhance their ability to withstand the external environment. The results demonstrate significant differences in cell fluorescence intensity between the high- and low-glucose culture medium treatment groups.

[0051] 3. Lipopolysaccharide sulfate (LPS) treatment and ATP level monitoring Lipopolysaccharide (LPS) Treatment: When iATPSnFR1 recombinant cardiomyocytes reached 70% confluence, they were treated with 0.5 μg / mL lipopolysaccharide (LPS). Untreated iATPSnFR1 recombinant cardiomyocytes served as a control group. LPS was diluted in serum-free DMEM medium and treated for 24 hours.

[0052] Fluorescence monitoring of ATP levels: During and after LPS treatment, changes in ATP levels in iATPSnFR1 recombinant cardiomyocytes were monitored in real time using a high-content imaging system with the same imaging parameters as above.

[0053] Data acquisition and analysis: A high-content imaging system was used to automatically acquire cell fluorescence images in multiple fields of view.

[0054] The results are as follows Figure 4 As shown in the figure, after AC16 cells were treated with 0.5 μg / mL LPS for 24 hours, the fluorescence intensity of iATPSnFR1 recombinant cardiomyocytes decreased significantly, indicating that LPS treatment can significantly reduce the ATP level in cardiomyocytes.

[0055] 3. Effects of different concentrations of lipopolysaccharide sulfate (LPS) on ATP levels in cardiomyocytes Cell confluence: Monitor the growth of AC16 cells. When the iATPSnFR1 recombinant cardiomyocytes reach 70% confluence, prepare for subsequent treatment to simulate the effects of lipopolysaccharide sulfate (LPS) on cellular ATP levels.

[0056] LPS treatment: After cells reach appropriate confluence, they are exposed to different concentrations of LPS (e.g., 0.5 μg / mL, 1.5 μg / mL, 2.5 μg / mL, and 3.5 μg / mL) for 24 hours to observe the effects of different LPS concentrations on ATP levels.

[0057] Fluorescence monitoring of ATP levels: During treatment, genetically encoded fluorescent sensors (such as iATPSnFRs) are used to monitor changes in intracellular ATP concentration in real time. This sensor can respond rapidly to changes in ATP concentration, showing a clear fluorescence change.

[0058] Fluorescence microscopy imaging: After treatment, image the cells using a fluorescence microscope. Select an appropriate excitation wavelength to capture the fluorescence signal of the iATPSnFRs sensor.

[0059] The present invention quantitatively evaluates the changes in ATP levels in different treatment groups using image analysis software. Figure 5 shown.

[0060] To more comprehensively analyze the overall changing trends of ATP levels in cardiomyocyte populations under different treatment conditions and verify the population representativeness of the results obtained by the high-content imaging system, we used flow cytometry to quantitatively analyze iATPSnFR1-recombined cardiomyocyte AC16 cells.

[0061] The specific steps for cell processing and flow cytometric analysis are as follows: Cell harvesting and washing: After different treatment times, the cardiomyocytes in the experimental and control groups were first harvested. For AC16 cells grown on the wall, the following steps were used for harvesting and washing: (1) Cell counting and concentration adjustment: After washing, the cell suspension was counted using a hemocytometer to accurately determine the cell concentration. According to the requirements of the flow cytometer, the cell concentration was adjusted to 1×10^6 cells / mL or other appropriate concentrations. Too high or too low a cell concentration will affect the analysis results of flow cytometry. (2) Cell filtration: To prevent cell aggregates from clogging the flow cytometer nozzle and affecting the accuracy of the data, the cell suspension needs to be filtered. Use a 300-mesh or 400-mesh cell screen or cell strainer to filter the cell suspension to remove cell clumps and debris to ensure that the flow cytometer detects a single cell suspension. (3) Flow cytometer detection: Use a flow cytometer (BD FACSCanto II flow cytometer) for fluorescence detection. (4) Flow data analysis: Use flow cytometer data analysis software to analyze the collected data.

[0062] Flow cytometry analysis results can be presented in a variety of formats, including: The detailed flow cytometric analysis steps described above enable quantitative and precise assessment of changes in cardiomyocyte ATP levels under different conditions. This provides population-level validation and complements findings from high-content imaging systems, thereby more comprehensively and reliably revealing the regulatory effects of various factors on cardiomyocyte energy metabolism. After centrifugation and resuspension, the cells were adjusted to a concentration suitable for flow cytometric analysis. The cell concentration was adjusted to 1×10^6 cells / mL to ensure that the flow cytometer could effectively capture the fluorescent signal of each cell.

[0063] Data analysis: Fluorescence microscopy and flow cytometry data were collected to analyze the effects of different concentrations of LPS on the ATP level of AC16 cells. By comparing the changes in ATP levels in different treatment groups, the results are as follows: Figure 6 As shown, LPS attenuated the fluorescence intensity of iATPSnFR1 recombinant cardiomyocytes.

[0064] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0065] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A method for dynamically monitoring ATP levels in myocardial cells, characterized in that: The iATPSnFRs gene shown in SEQ ID NO.3 was cloned into the multiple cloning site of the pLVX-AcGFP1-N1 plasmid to construct the iATPSnFR1 recombinant plasmid; The iATPSnFR1 recombinant plasmid was transformed into Escherichia coli, and positive bacterial clones containing the iATPSnFR1 recombinant plasmid were screened. The plasmid was extracted and transfected into cardiomyocytes to obtain iATPSnFR1 recombinant cardiomyocytes; Monitor changes in ATP levels in iATPSnFR1 recombinant cardiomyocytes.

2. The method for dynamically monitoring ATP levels in myocardial cells according to claim 1, characterized in that: The amplification primers for the iATPSnFRs gene include iATPSnFR1-F shown in SEQ ID NO.1 and iATPSnFR1-R shown in SEQ ID NO.

2.

3. The method for dynamically monitoring ATP levels in myocardial cells according to claim 1, wherein: iATPSnFRs gene was cloned into pLVX-AcGFP1-N1 plasmid Bam HI and Xba I site.

4. The method for dynamically monitoring ATP levels in myocardial cells according to claim 1, wherein: The confluence of cardiomyocytes was 70% to 80%.

5. An iATPSnFR1 recombinant plasmid, characterized in that: The iATPSnFRs gene shown in SEQ ID NO.3 of claim 1 was cloned into the pLVX-AcGFP1-N1 plasmid. Bam HI and Xba I site, and the iATPSnFR1 recombinant plasmid was constructed.

6. A recombinant cardiomyocyte, characterized in that: The method is obtained by transfecting myocardial cells with the iATPSnFR1 recombinant plasmid according to claim 5.

7. The recombinant cardiomyocyte according to claim 6, characterized in that The cardiomyocytes are AC16 cardiomyocytes.

8. Use of the iATPSnFR1 recombinant plasmid according to claim 5 or the recombinant cardiomyocyte according to claim 6 in dynamically monitoring ATP levels in cardiomyocytes.

9. The use according to claim 8, characterized in that A high-content imaging system was used to monitor changes in ATP levels in iATPSnFR1-recombinant cardiomyocytes in real time.

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