A real-time monitoring method for myocardial injury caused by long-term coronary pneumonia based on a biosensor system

Abstract

The present invention relates to a real-time monitoring method for long-term coronary myocardial injury based on a biosensor system. The method mainly monitors the electrophysiological characteristics of myocardial cells under the infection of the new coronavirus pseudovirus in real time through a multi-electrode array module, and combines the optical signal live cell imaging technology to detect the dynamic changes of calcium ions and cell survival rate and apoptosis. Through this method, researchers can simulate the infection process of long-term coronavirus on myocardial cells in an in vitro system, and deeply analyze the electrophysiological abnormalities, calcium ion influx regulation and cell damage mechanisms of myocardial cells caused by the virus. The core technologies of this invention include electrophysiological signal acquisition based on MEA and calcium ion flow monitoring based on optical signals. The results of the study showed that this model method can effectively reveal the cytological mechanism of long-term coronary myocardial injury, provide a powerful tool for the basic research of new coronavirus-related cardiovascular diseases, and lay a solid foundation for the subsequent drug screening and development of treatment plans.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and in particular to a method for real-time monitoring of myocardial injury caused by prolonged COVID-19 based on a biosensor system. Background Technology

[0002] Acute sequelae of SARS-CoV-2 (COVID-19) infection (PASC) manifest as persistent symptoms following the initial stages of COVID-19 infection, affecting an estimated 10-30% of survivors worldwide. These symptoms significantly impact cardiovascular health, leading to myocardial damage and arrhythmias, drastically reducing patients' quality of life. Existing diagnostic techniques, such as echocardiography and cardiac magnetic resonance imaging, cannot reveal the disease's effects at the high-resolution cellular level; therefore, the underlying mechanisms of cardiac dysfunction caused by PASC remain unclear, hindering the development of corresponding effective treatments. Therefore, establishing an efficient and practical in vitro platform for studying myocardial damage induced by PASC is crucial for advancing the development of precise diagnostic and therapeutic strategies.

[0003] Pseudoviruses, as engineered viruses, mimic the SARS-CoV-2 infection process by expressing spike proteins, but lack the ability to replicate and cause disease. Pseudoviruses facilitate the safe exploration of the effectiveness of various antibodies or serological mechanisms in inhibiting viral entry, thereby accelerating the development and evaluation of COVID-19 vaccines. Furthermore, pseudoviruses are crucial for elucidating the dynamic processes of viral entry into host cells and studying the molecular interactions between viral spike proteins and host receptors such as ACE2; these insights are fundamental for developing precise interventions to slow the spread of infection. Pseudoviruses also play a vital role in drug discovery, serving as an important tool for high-throughput screening of antiviral agents. Their ability to facilitate the discovery of compounds with potential viral entry-inhibiting effects accelerates the advancement of treatments to clinical trials. With their nanoscale size, pseudoviruses provide a versatile platform at the intersection of nanotechnology and virology to advance research on viral pathogenic mechanisms and prepare for future pandemics.

[0004] To elucidate the impact of COVID-19 on the cardiovascular system, cutting-edge clinical and experimental methods play a crucial role. Clinically, imaging and specific biomarker detection techniques can rigorously assess myocardial function and damage in infected patients. However, these conventional tools are insufficient to capture the persistent pathophysiological dysfunction that occurs during infection. To address these clinical challenges, complex in vitro systems utilizing primary cardiomyocytes and cardiomyocyte lines are essential for studying the direct cardiac effects of SARS-CoV-2 infection. These systems reveal the mechanisms by which the virus induces inflammation and myocardial injury, but often fail to reproduce the complex, specific responses and chronic manifestations of PASCs. Long-duration dynamic microelectrode array (MEA) recording of cardiomyocyte electrophysiological activity can compensate for the data gaps in conventional detection methods. By providing persistent, high-resolution insights into action potentials and arrhythmia patterns, MEA technology significantly advances a comprehensive understanding of the long-term adaptation of cardiomyocytes to viral effects and therapeutic interventions. This approach is crucial for studying persistent cardiac lesions and designing targeted treatment regimens. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing in vitro monitoring methods for virus-induced myocardial injury in terms of long-term dynamic monitoring and multi-dimensional data acquisition, by providing a real-time monitoring method for prolonged COVID-19-induced myocardial injury based on a biosensor system. This method can monitor the electrophysiological characteristics and dynamic changes in calcium ions of cardiomyocytes over a long period, thereby achieving a comprehensive assessment of myocardial injury after prolonged COVID-19 infection and can be used for efficacy studies of antiarrhythmic drugs.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for real-time monitoring of prolonged COVID-19-induced myocardial injury based on a biosensor system, the method comprising the following steps:

[0008] (1) In vitro culture of cardiomyocytes on a multi-electrode array (MEA): Cardiomyocytes were seeded on the MEA chip and cultured until the cells adhered to the wall and began to beat spontaneously.

[0009] (2) SARS-CoV-2 pseudovirus infection: SARS-CoV-2 pseudovirus expressing the spike protein was added to the myocardial cell culture medium to simulate the viral infection process;

[0010] (3) Electrophysiological signal monitoring: The electrophysiological signals of myocardial cells, including changes in action potential and electric field potential, are recorded in real time through the MEA module;

[0011] (4) Calcium ion flow monitoring: Using optical signal live cell imaging technology and calcium ion fluorescent probes, the dynamic changes in intracellular calcium ion concentration are monitored in real time.

[0012] (5) Dynamic monitoring: Dynamically monitor the changes in electrophysiological signals and calcium ion flow of cardiomyocytes after viral infection for N days, with N>5, in order to capture the dynamic process of chronic myocardial injury caused by COVID-19 and to assess cardiomyocyte activity.

[0013] Specifically, in step (1), the cardiomyocytes are primary cardiomyocytes or cardiomyocytes differentiated from induced pluripotent stem cells (iPSCs).

[0014] Specifically, the pseudoviruses in step (2) include lentiviruses expressing the SARS-CoV-2 spike protein and its variants, used to simulate SARS-CoV-2 infection and can be used to screen and evaluate the cardiotoxicity of anti-SARS-CoV-2 drugs.

[0015] Specifically, in step (2), the pseudovirus can be adjusted in concentration to simulate different levels of viral infection, with a concentration range of 10. 4 Up to 10 9 IU / ml.

[0016] Specifically, the MEA module in step (3) contains 8 to 5000 electrodes with a diameter of 10 to 30 μm and a distribution density of 100 to 1000 electrodes / cm². 2 It is used to simultaneously monitor the electrical activity of several myocardial cells.

[0017] Specifically, the optical signal live-cell imaging technology uses a calcium ion fluorescent probe, which emits a fluorescent signal by binding with calcium ions, thereby monitoring the dynamic changes in calcium ion release. The monitoring of the dynamic changes in calcium ion influx is performed using a high-sensitivity fluorescence microscope with an imaging speed of more than 10 frames per second to capture the dynamic changes in intracellular calcium ions. The calcium ion fluorescent probe includes, but is not limited to, the following four types: Fura-2 AM, Fluo-4 AM, Rhod-2 AM, and Calcium Green-1 AM.

[0018] Furthermore, the assessment of cardiomyocyte viability includes labeling apoptotic and surviving cells with Annexin-V and DAPI fluorescent dyes and analyzing them using flow cytometry.

[0019] Specifically, the fake virus 0-5×10 7 Assess the dose-response relationship of viral damage to cardiomyocytes within the IU dose range.

[0020] Specifically, the monitoring process in step (5) is carried out under standard culture conditions, maintaining a temperature of 37°C and a CO2 concentration of 5%.

[0021] Furthermore, based on viral infection of cardiomyocytes, the effects of antiarrhythmic drugs on cardiomyocyte electrophysiological abnormalities can be evaluated to assess their cardioprotective effects. Changes in cardiomyocyte electrical activity and calcium ion dynamics before and after drug treatment can be analyzed using MEA and calcium ion flow monitoring to quantify the protective effect of antiarrhythmic drugs. The antiarrhythmic drugs include, but are not limited to, the following four: nifedipine, amiodarone, propranolol, and lidocaine.

[0022] The beneficial effects of this invention are: the method of this invention is simple to operate and can be used for long-term dynamic monitoring of the physiological changes of cardiomyocytes under SARS-CoV-2 pseudovirus infection, which is beneficial for a comprehensive assessment of the virus's impact on cardiomyocytes. This invention provides a real-time monitoring model method for long-term COVID-19-related myocardial injury based on a multimodal biosensor system, which can acquire high-quality electrophysiological signals of cardiomyocytes and dynamic changes in calcium ions, and can extend the monitoring time, thereby achieving accurate and long-term monitoring of cardiomyocyte function. This provides an important experimental platform and technical support for research on long-term COVID-19-related cardiac lesions and drug development. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0024] Figure 1 This is a diagram of the in vitro culture of cardiomyocytes in a biosensor system according to the present invention;

[0025] Figure 2 This is a diagram of primary cardiomyocytes infected with the SARS-CoV-2 pseudovirus of this invention;

[0026] Figure 3 This invention presents a detection diagram of electrical signals and calcium ion flow in primary cardiomyocytes infected with SARS-CoV-2 pseudovirus over a long period of time.

[0027] Figure 4 This is a detection graph of electrical signals and calcium ion flow of nifedipine against SARS-CoV-2 pseudovirus infection according to the present invention. Detailed Implementation

[0028] This invention provides a real-time monitoring model method for long-term COVID-19-induced myocardial injury based on a multimodal biosensor system, comprising: seeding cardiomyocytes on a multi-electrode array (MEA) chip for culture; infecting cardiomyocytes with a pseudovirus expressing the SARS-CoV-2 spike protein; monitoring the electrophysiological signals and dynamic changes in calcium ion flow of cardiomyocytes; and evaluating the protective effect of antiarrhythmic drugs on cardiomyocyte dysfunction (cardiomyocyte viability assessment includes labeling apoptotic and surviving cells with Annexin-V and DAPI fluorescent dyes, and analyzing the results using flow cytometry; obtaining cell apoptosis rate and survival rate). This model method is simple to operate, can be used for long-term dynamic monitoring of the physiological changes of cardiomyocytes under viral infection, and can systematically evaluate the effectiveness of antiarrhythmic drugs. The invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0029] A real-time monitoring model method for long-term COVID-19-induced myocardial injury based on a multimodal biosensor system includes the following steps:

[0030] (1) In vitro culture of cardiomyocytes on MEA:

[0031] Cardiac cells were seeded on MEA chips and cultured until they adhered and began to beat spontaneously, ensuring a stable cell state.

[0032] Furthermore, the cardiomyocytes may be primary cardiomyocytes or cardiomyocytes differentiated from induced pluripotent stem cells (iPSCs).

[0033] (2) SARS-CoV-2 pseudovirus infection:

[0034] Pseudoviruses expressing the SARS-CoV-2 spike protein were added to the culture medium of cardiomyocytes to simulate the viral infection process.

[0035] Furthermore, the concentration of the pseudovirus is adjustable to simulate different levels of viral infection, with a commonly used concentration range of 10. 4 Up to 10 9 IU / ml.

[0036] (3) Monitoring of electrophysiological signals:

[0037] The MEA module records the electrophysiological signals of cardiomyocytes in real time, including changes in action potential and electric field potential.

[0038] Furthermore, the MEA module comprises 8 to 5000 electrodes with a diameter of 10 to 30 μm and a distribution density of 100 to 1000 electrodes / cm². 2 It is used to simultaneously monitor the electrical activity of multiple myocardial cells.

[0039] (4) Calcium ion flow monitoring:

[0040] Using optical signal live-cell imaging technology and a calcium ion fluorescent probe, the dynamic changes in intracellular calcium ion concentration in cardiomyocytes can be monitored in real time.

[0041] The calcium ion fluorescent dyes used include, but are not limited to:

[0042] Fura-2 AM: A widely used ratiometric calcium ion dye that can accurately measure intracellular calcium ion concentration via UV excitation.

[0043] Fluo-4 AM: A highly sensitive calcium ion dye that is excited by visible light, suitable for high-throughput screening and real-time calcium imaging.

[0044] Rhod-2 AM: A red fluorescent calcium ion dye with high photostability, suitable for detecting changes in calcium ions within mitochondria.

[0045] Calcium Green-1 AM: A calcium ion dye with high sensitivity and good photostability, suitable for long-term calcium imaging experiments.

[0046] Furthermore, the monitoring of calcium ion flow was performed using a high-sensitivity fluorescence microscope with an imaging speed of more than 10 frames per second to capture rapid dynamic changes in intracellular calcium ions.

[0047] (5) Long-term dynamic monitoring:

[0048] This method captures the dynamic process of chronic myocardial injury induced by long-term (5 days or more) dynamic monitoring of changes in electrophysiological signals and calcium ion flow in cardiomyocytes after viral infection.

[0049] Furthermore, the monitoring process can be carried out under standard culture conditions, maintaining a temperature of 37°C and a CO2 concentration of 5%.

[0050] Evaluation of antiarrhythmic drugs:

[0051] Based on viral infection of cardiomyocytes, this study investigates the effects of commonly used antiarrhythmic drugs on cardiomyocyte electrophysiological abnormalities and evaluates their cardioprotective effects. The antiarrhythmic drugs studied include, but are not limited to:

[0052] Nifedipine: A calcium channel blocker widely used to treat hypertension and angina pectoris, it reduces electrophysiological abnormalities of cardiomyocytes by inhibiting calcium ion influx.

[0053] Amiodarone: A broad-spectrum antiarrhythmic drug that prevents and treats arrhythmias by prolonging the action potential duration and reducing myocardial excitability.

[0054] Propranolol: A non-selective beta-blocker that prevents arrhythmias by lowering heart rate and reducing myocardial oxygen consumption.

[0055] Lidocaine: A sodium channel blocker that reduces the conduction of abnormal electrical signals by inhibiting the influx of sodium ions and is often used to treat acute arrhythmias.

[0056] Furthermore, changes in cardiomyocyte electrical activity and calcium ion dynamics before and after drug treatment were analyzed using MEA and calcium ion flow monitoring to quantify the protective effect of the drug.

[0057] Example 1:

[0058] A real-time monitoring model method for long-term COVID-19-induced myocardial injury based on a multimodal biosensor system includes the following steps:

[0059] (1) In vitro culture of cardiomyocytes on MEA:

[0060] like Figure 1 As shown, the MEA device is primarily manufactured using photolithography. The substrate of the device is made of four-inch quartz glass. First, a 2.5μm thick layer of positive photoresist is sprayed onto the glass substrate at 3000rpm. After soft baking at 120℃ for two minutes, it is developed for 35 seconds using RZX3038 developer, and then aligned through a mask at 300mJ / cm². 2 Intensity exposure was used to form conductive design patterns. A 10nm titanium / 100nm gold layer was deposited on the substrate using magnetron sputtering, followed by acetone stripping of the photoresist. Next, a 5μm thick layer of SU-8 2005 photoresist was spin-coated at 3000 rpm and soft-baked at 95°C for two minutes. Afterwards, a mask aligner was used at 160mJ / cm². 2 The SU-8 layer was exposed to a high intensity and then post-exposed and baked at 95°C for three minutes. After one minute of development, the layer was rinsed with propylene glycol methyl ether acetate (PGMEA), then washed with isopropanol, dried with a nitrogen torch, and baked at 150°C for 30 minutes. Finally, the substrate was cut into 25 pieces of 2×2cm. 2Each chip contains 32 microelectrodes. The microelectrode array chip was then attached to a custom-designed printed circuit board (PCB) adapter using polydimethylsiloxane (PDMS). Conductive silver paste was used to electrically connect the electrode pads on the chip to the corresponding pads on the PCB. Glass rings serving as cell culture pools were fixed to the chip using PDMS. The PCB was equipped with pin connectors for interfacing with a self-developed biosensing system. The device was sterilized in a biosafety cabinet after rinsing with 75% ethanol and then irradiated with ultraviolet light for 2 hours. Before cell seeding, the device was coated with a 10 μg / mL fibronectin solution and incubated overnight at 4°C.

[0061] Primary neonatal cardiomyocytes were obtained from 1-day-old Wistar rats. After disinfection with 75% ethanol, the heart tissue was rapidly excised and placed in Hanks' balanced salt solution (HBSS), followed by rinsing with ice-cold Dulbecco modified Eagle medium (DMEM). The ventricular tissue was then cut into approximately 1 mm sections. 3 Small pieces of the cells were digested in HBSS containing 0.05% type II collagenase and 0.07% trypsin. The digestion process was repeated ten to twelve times in a 37°C, 5% CO2 incubator. After each digestion, the supernatant was collected and centrifuged at 1000 rpm for 5 minutes. Cardiomyocytes were purified by differential adhesion, and the purification process was repeated twice to improve purity. The isolated cardiomyocytes were then resuspended in DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and subsequently seeded onto MEA devices. To maintain optimal growth conditions, the culture medium was changed every 48 hours.

[0062] (2) SARS-CoV-2 pseudovirus infection:

[0063] Will Figure 1 Neonatal rat cardiomyocytes from the Middle Plain generation were cultured and subjected to SARS-CoV-2 pseudovirus expressing GFP (7.26 × 10⁻⁶). 8 Infection was performed using IU / ml, 5 μL / well, 10 μL / well, and 20 μL / well at optimized MOI (multiple of infection). To promote viral entry into cells, 10 μg / mL polybrominated compound was added. Cells were then incubated at 37°C and 5% CO2 for 48 hours. GFP expression in infected cardiomyocytes was observed using a fluorescence microscope, and images were captured. Figure 2 The image shows the detection of GFP expression in primary cardiomyocytes after infection with SARS-CoV-2 pseudovirus. The figure illustrates that the pseudovirus can enter primary cardiomyocytes, and GFP expression is dose-dependent 48 hours after infection.

[0064] (1) Long-term dynamic monitoring of electrical signals and calcium ion flow

[0065] Extracellular action potentials of cardiomyocytes are transmitted through Figure 1 The customized biosensing system shown is used for recording, and this system integrates electroporation and signal recording functions, such as... Figure 3 As shown. First, the primary amplifier module captures and amplifies the weak electrical signals in the device tenfold, then transmits them through signal conditioning circuitry designed for precise electrophysiological recording. These signals are then further amplified fiftyfold by a secondary amplifier and sampled at a frequency of 20 kHz by an analog-to-digital converter (ADC) within a high-speed data acquisition (DAQ) card. To reduce noise, a bandpass filter from 1 Hz to 7.5 kHz was used. The DAQ card has a detection range of -5 to +5 V, and the ADC provides 16-bit resolution with a sensitivity of 153 μV. The operation of the entire system, including signal recording via USB communication, and subsequent data presentation, acquisition, storage, and processing, is controlled by a custom-developed LabVIEW program. All cell experiments were performed in a controlled environment of 37°C and 5% CO2 to ensure conditions suitable for long-term monitoring. Two days after cell seeding into the device, cells were infected with SARS-CoV-2 pseudovirus. Extracellular electrical signals from cells not infected with pseudovirus were initially recorded to establish a baseline, followed by continuous recording of signals over five days after infection.

[0066] Calcium ion flow detection: Treated cardiomyocytes were cultured in 24-well plates. A 5 mM stock solution was prepared by dissolving Rhod-4 AM dye in DMSO. Cells were washed with HBSS and incubated for 30 min at 37°C in HBSS containing 2 μM Rhod-4 AM working solution, protected from light. After incubation, cells were washed with HBSS and allowed to stand for 30 min to stabilize. Fluorescence imaging was then performed using a fluorescence microscope. Changes in fluorescence intensity were recorded to monitor calcium ion flow in real time.

[0067] pass Figure 3 The monitoring of electrical and optical signals shown indicates the establishment of a long-term dynamic monitoring model for myocardial injury caused by COVID-19, laying the foundation for a systematic evaluation of the effectiveness of antiarrhythmic drugs.

[0068] Example 2:

[0069] Evaluation of antiarrhythmic drugs. Use. Figure 1Primary neonatal rat cardiomyocytes were first cultured and stabilized to spontaneous beating on a MEA chip. Then, 10 μL of SARS-CoV-2 pseudovirus was added to infect the cardiomyocytes to simulate chronic myocardial injury associated with long-term COVID-19. Simultaneously with viral infection, the experimental group was treated with 100 nM nifedipine, and the electrophysiological activity and calcium ion flow of cardiomyocytes were monitored in real time over the next five days. The MEA module recorded the action potential amplitude and firing frequency of the cardiomyocytes, and intracellular calcium ions were labeled using Rhod-4 AM dye. Dynamic changes in calcium ions were observed using fluorescence microscopy. Furthermore, morphological evaluation was performed to analyze the effects of nifedipine on cardiomyocyte morphology and viability. Based on the combined results of electrophysiological signals, calcium ion dynamics, and morphological evaluation, nifedipine showed significant cardioprotective effects in inhibiting SARS-CoV-2 pseudovirus-induced arrhythmias and restoring calcium ion homeostasis, particularly in the early and late stages after infection, demonstrating strong therapeutic potential.

[0070] Figure 4 This image shows the electrophysiological signal and calcium ion flow changes of nifedipine in suppressing arrhythmias, as monitored in this case study. Morphological analysis was used to comprehensively evaluate the efficacy of nifedipine against myocardial injury caused by prolonged COVID-19.

[0071] In summary, the above embodiments are merely illustrative of the principles and effects of the present invention and do not constitute a limitation on the implementation methods. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and scope of the present invention, and all such modifications and improvements should be considered within the scope of protection of the present invention.

Claims

1. A method for real-time monitoring of prolonged COVID-19-induced myocardial injury based on a biosensor system, characterized in that, The method is implemented through the following steps: (1) In vitro culture of cardiomyocytes on a multi-electrode array (MEA): Cardiomyocytes were seeded on the MEA chip and cultured until the cells adhered to the wall and began to beat spontaneously. (2) SARS-CoV-2 pseudovirus infection: SARS-CoV-2 pseudovirus expressing the spike protein was added to the myocardial cell culture medium to simulate the viral infection process; (3) Electrophysiological signal monitoring: The electrophysiological signals of myocardial cells, including changes in action potential and electric field potential, are recorded in real time through the MEA module; (4) Calcium ion flow monitoring: Using optical signal live cell imaging technology and calcium ion fluorescent probes, the dynamic changes in intracellular calcium ion concentration are monitored in real time. (5) Dynamic monitoring: Dynamically monitor the changes in electrophysiological signals and calcium ion flow of cardiomyocytes after viral infection for N days, with N>5, in order to capture the dynamic process of chronic myocardial injury caused by COVID-19 and to assess cardiomyocyte activity.

2. The method according to claim 1, characterized in that, In step (1), the cardiomyocytes are primary cardiomyocytes or cardiomyocytes differentiated from induced pluripotent stem cells (iPSCs).

3. The method according to claim 1, characterized in that, The pseudoviruses in step (2) include lentiviruses expressing the SARS-CoV-2 spike protein and its variants, used to simulate SARS-CoV-2 infection and to screen and evaluate the cardiotoxicity of anti-SARS-CoV-2 drugs.

4. The method according to claim 1, characterized in that, In step (2), the pseudovirus can be adjusted in concentration to simulate different levels of viral infection, with a concentration range of 10. 4 Up to 10 9 IU / ml.

5. The method according to claim 1, characterized in that, The MEA module in step (3) contains 8 to 5000 electrodes with a diameter of 10 to 30 μm and a distribution density of 100 to 1000 electrodes / cm². 2 It is used to simultaneously monitor the electrical activity of several myocardial cells.

6. The method according to claim 1, characterized in that, The optical signal live-cell imaging technology uses a calcium ion fluorescent probe, which emits a fluorescent signal by binding with calcium ions, thereby monitoring the dynamic changes in calcium ion release. The monitoring of the dynamic changes in calcium ion influx is performed using a high-sensitivity fluorescence microscope with an imaging speed of more than 10 frames per second to capture the dynamic changes in intracellular calcium ions. The calcium ion fluorescent probe includes, but is not limited to, the following four types: Fura-2AM, Fluo-4 AM, Rhod-2 AM, and Calcium Green-1 AM.

7. The method according to claim 1, characterized in that, The assessment of cardiomyocyte viability included labeling apoptotic and surviving cells with Annexin-V and DAPI fluorescent dyes, followed by analysis using flow cytometry.

8. The method according to claim 1, characterized in that, The fake virus 0-5×10 7 Assess the dose-response relationship of viral damage to cardiomyocytes within the IU dose range.

9. The method according to claim 1, characterized in that, The monitoring process in step (5) is carried out under standard culture conditions, maintaining a temperature of 37°C and a CO2 concentration of 5%.

10. The method according to claim 1, characterized in that, Based on viral infection of cardiomyocytes, the effects of antiarrhythmic drugs on cardiomyocyte electrophysiological abnormalities can be evaluated, and their cardioprotective effects can be assessed. Changes in cardiomyocyte electrical activity and calcium ion dynamics before and after drug treatment can be analyzed using MEA and calcium ion flow monitoring to quantify the protective effect of antiarrhythmic drugs. The antiarrhythmic drugs include, but are not limited to, the following four: nifedipine, amiodarone, propranolol, and lidocaine.

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