A method for screening drugs with cardiac ion channel toxicity

Abstract

This invention relates to the field of drug safety testing technology, specifically to a method for screening drugs with cardiac ion channel toxicity. The method includes: infecting cardiomyocytes with a lentiviral system carrying the SV40 large T antigen to cultivate a conditional cardiomyocyte cell line; labeling the conditional cardiomyocyte cell line with a voltage-sensitive dye or a calcium ion-sensitive dye; adding the drug to be tested and a buffer solution to a culture dish containing the labeled conditional cardiomyocytes to form a test group and a control group; performing optical mapping on the test group and the control group, and statistically analyzing the optical voltage mapping results; wherein, in the optical mapping, the electrical stimulation method is point stimulation; the stimulation parameters are: pulse width 1-3 ms, voltage amplitude 6-9 V, and frequency 1-10 Hz. The method provided by this invention does not require complex treatment with chemical substances and can perform artifact-free and synchronized electrical stimulation of cardiomyocytes within the physiological heart rate range, making it suitable for high-throughput cardiotoxicity screening experiments.

Description

Technical Field

[0001] This invention relates to the field of drug safety testing technology, specifically to a method for screening drugs with cardiac ion channel toxicity. Background Technology

[0002] Cardiac action potentials (APs) are formed by the sequential opening and closing of specific ion channels. In human ventricular wall cardiomyocytes, the initial rapid depolarization is initiated by the Na+ ionization of SCN5a (Nav1.5) channels. + Electrical conduction is crucial for the rapid propagation of electrical activity within the heart. Subsequently, the AP platform consists of an L-type Ca... 2+ The channel remains open, Ca 2+ Inflow causes intracellular Ca to be stored 2+ Release to initiate contraction. Human AP is caused by Ca 2+ Channel closure and activation termination of two repolarizing potassium channels, namely the fast activation delayed rectified current (IKr) of the hERG channel and the slow activation component (IKs) generated by the Kv7.1 channel, constitute the "repolarization reserve". The resting membrane potential between APs is maintained by the inward rectified potassium current (IK1) of the Kir2.1 channel.

[0003] However, hereditary ion channel mutations, drug side effects, or electrolyte imbalances can affect the activity of these channels, altering the morphology of acute pancreatic tachycardia (AP) and posing a high risk of potentially fatal ventricular tachycardia. Common cardiotoxic effects of drugs include long QT syndrome (LQTS), short QT syndrome (SQTS), and Brugada syndrome (BS).

[0004] To reduce post-marketing cardiac safety risks, the U.S. Food and Drug Administration (FDA) mandates that all new drugs undergo hERG channel inhibition testing before entering clinical trials. This screening method aims to determine whether the drug will induce QT interval prolongation or cause fatal arrhythmias (such as torsades de pointes). However, this screening method only detects a single hERG potassium channel, leading to serious false positive and false negative problems: approximately 30% of drugs without cardiotoxicity are incorrectly rejected due to hERG channel inhibition, resulting in a waste of research and development resources; and some drugs that pass the hERG screening may still cause arrhythmias in clinical trials or after market launch, failing to effectively guarantee the drug's cardiac safety.

[0005] In-depth analysis reveals that existing drug cardiotoxicity screening technologies have many shortcomings, mainly concentrated in two aspects: cell models and detection techniques. Regarding cell models, existing models fail to meet the demands for efficient, accurate, and low-cost drug screening: primary cardiomyocytes are difficult to obtain, making it hard to meet the quantity requirements for high-throughput screening; while human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) exhibit some similarity in ion channel expression to primitive human cardiomyocytes, they suffer from high cellular heterogeneity and low maturity, making them unsuitable for arrhythmia detection, and their high culture cost prevents large-scale application; the HL-1 cell line suffers from slow conduction velocity (only 1 cm / s) and abnormal action potential morphology, failing to accurately simulate the electrophysiological characteristics of normal cardiomyocytes; furthermore, obtaining animal cardiomyocytes is time-consuming, labor-intensive, and costly, and sufficient quantities of primitive human cardiomyocytes cannot be obtained through biopsy for drug screening, and heterologous expression of hERG K... + The cell line of the channel lacks other key Na+ in cardiomyocytes. + Ca 2+ or K + The channel cannot truly simulate the effect of drugs on cardiomyocytes, thus limiting the accuracy of screening.

[0006] In terms of detection technology, existing technologies all have obvious limitations, making it difficult to achieve efficient and accurate cardiotoxicity screening: Patch-clamp technology, as the gold standard for studying the electrical properties of cardiomyocytes, has extremely low throughput, only able to test a few samples per day, which cannot meet the high-throughput screening needs in the process of new drug development. Moreover, the duration of the experiment is limited by the consumption of cytoplasmic stability, further reducing the screening efficiency. Microelectrode array (MEA) technology suffers from serious stimulation artifacts, which can lead to the inaccurate recording of the rapid depolarization phase of the action potential. In addition, toxic gases are generated during stimulation and the environmental pH is changed. Furthermore, the shape of its fluorescence signal (FP) varies between individual electrodes, requiring investigators to manually select the appropriate electrode, which hinders the realization of automated analysis. In addition, most cardiomyocyte studies on MEAs can only be carried out during spontaneous cell beating, while the effects of drugs on cardiac excitability and action potentials are often related to heart rate, which affects the accuracy of screening results.

[0007] In summary, existing drug cardiotoxicity screening technologies suffer from problems such as unreasonable cell models, defective detection techniques, low screening accuracy, insufficient throughput, and high costs, failing to meet the needs of efficient, accurate, and low-cost cardiotoxicity screening in new drug development. Therefore, developing a drug cardiotoxicity screening technology that can overcome the above-mentioned defects has significant practical significance and application value. Summary of the Invention

[0008] Based on this, the purpose of the present invention is to provide a method for screening cardiac ion channel toxic drugs, so as to solve the problems of existing detection technologies being unable to be used on a large scale, having low automation, interference from stimuli, limited experimental scenarios, low experimental efficiency, and long experimental time.

[0009] To achieve the above objectives, the present invention adopts the following technical solution.

[0010] This invention provides a method for screening drugs with cardiac ion channel toxicity, comprising: infecting cardiomyocytes with a lentiviral system carrying the SV40 large T antigen to cultivate a conditional cardiomyocyte cell line; The conditional cardiomyocyte lines were labeled using voltage-sensitive dyes or calcium ion-sensitive dyes. The drug to be tested and the buffer solution were added to a culture dish containing labeled conditional cardiomyocytes to form a test group and a control group. Optical voltage calibration was used to test the test group and the control group, and the results of the optical voltage calibration were analyzed for statistical significance. In the optical voltage mapping, the electrical stimulation method is point stimulation; the stimulation parameters are: pulse width 1-3 ms, voltage amplitude 6-9 V, and frequency 1-10 Hz. The electrical stimulation parameters are set within this range to achieve an intensity and width sufficient to effectively stimulate the conditional cardiomyocyte line to generate action potentials without causing cell damage. Preferably, the stimulation parameters are: pulse width 2 ms and voltage amplitude 8 V.

[0011] In some technical solutions of the present invention, the drug to be tested includes at least one of a voltage-gated sodium channel Nav1.5 blocker, a voltage-gated potassium channel Kv11.1 / hERG blocker, an L-type calcium channel Cav1.2 agonist, and an L-type calcium channel Cav1.2 blocker.

[0012] In some preferred embodiments of the present invention, the voltage-gated sodium channel Nav1.5 blocker includes tetrodotoxin; the voltage-gated potassium channel Kv11.1 / hERG blocker includes astemizole; the L-type calcium channel Cav1.2 agonist includes BayK8644; and the L-type calcium channel Cav1.2 blocker includes nifedipine.

[0013] In some technical solutions of the present invention, the optical voltage detection results include: the amplitude, duration, and conduction velocity of the action potential of the test group and the control group.

[0014] In some technical solutions of the present invention, the optical voltage detection results include: action potential amplitude, action potential duration, conduction velocity, calcium transient amplitude, and calcium transient duration of the test group and the control group.

[0015] In some technical solutions of this invention, the conduction velocity of the voltage-gated sodium channel Nav1.5 blocker is significantly lower than that of the control group; the action potential durations (APD40 and APD90) of the voltage-gated potassium channel Kv11.1 / hERG blocker are significantly increased; the calcium flux amplitude and duration of the L-type calcium channel Cav1.2 agonist are significantly increased, and the calcium flux amplitude and duration of the L-type calcium channel Cav1.2 blocker are significantly decreased. In this invention, when there is a significant difference, P < 0.05.

[0016] In some technical solutions of this invention, the SV40 large T antigen is a temperature-sensitive mutant tsA58.

[0017] In some technical solutions of this invention, the lentiviral system employs the Tet-on / tTS tetracycline-induced expression system to achieve doxycycline-dependent expression of the large T antigen.

[0018] In some technical solutions of the present invention, the expression of the large SV40 large T antigen is driven by a myocardial specific promoter.

[0019] In some technical solutions of this invention, the myocardial-specific promoter is the MHCK7 promoter.

[0020] In some technical solutions of the present invention, a lentiviral system carrying the SV40 large T antigen is used to infect cardiomyocytes in a culture medium containing doxycycline.

[0021] In some technical solutions of this invention, the multiplicity of infection (MOI) of cardiomyocytes infected with a lentiviral system carrying the SV40 large T antigen is 1 to 10.

[0022] Based on the technical solution of the present invention, the present invention has the following beneficial effects compared with the prior art: The method for screening cardiac ion channel toxicity drugs provided by this invention utilizes a cell line that spontaneously differentiates into cardiomyocytes with electrical and mechanical activity, phenotypically similar to neonatal rat atrial myocytes. This eliminates the need for expensive biochemical reagents and complex treatment protocols, providing an easy-to-use and cost-effective system for addressing specific basic and applied cardiac research problems. It is an ideal model for studying the regulatory activity of toxins and drugs on cardiac ion channels. Furthermore, this invention incorporates optical voltage mapping technology, enabling artifact-free and synchronized electrical stimulation of cardiomyocytes within the physiological heart rate range. This avoids the inherent limitations of existing MEA and patch-clamp techniques, allowing for more precise characterization of the electrophysiological effects of drugs within the range of cardiac physiological activity. It also allows for averaging and objective quantitative analysis of action potentials. This screening method can screen hundreds of compounds per day, with a throughput far exceeding traditional patch-clamp detection techniques, making it highly suitable for large-scale, high-throughput screening of cardiac toxicity in the early stages of new drug development. Attached Figure Description

[0023] Figure 1 The diagram shows the results of lentivirus-mediated induction of SV40 large T antigen (LT) expression in primary atrial myocytes; among them, Figure 1 In this context, A represents a Western blot band plot; Figure 1 In this context, B represents the quantitative statistical result of the grayscale of the Western blot strip image.

[0024] Figure 2 This is an electrophysiological verification diagram of the differentiation and maturation of the conditional cardiomyocyte line (iAM); among which, Figure 2 In the diagram, A represents the typical activation map (isochronous interval 6 ms) and light signal trajectory map of iAM cells at 5 time points (days 0, 1, 2, 3, and 9) during differentiation, detected by optical mapping. Figure 2 The figure shows the statistical analysis results comparing the conduction velocity (CV) of iAM cells with that of primary atrial myocytes (pAMs) on days 3 and 9 of differentiation. Figure 2 The figure shows the statistical analysis results comparing the 30% repolarization time (APD30) of iAM cells with primary atrial myocytes (pAMs) on days 3 and 9 of differentiation. Figure 2 The figure shows the statistical analysis results comparing the 80% repolarization time (APD80) of iAM cells with that of primary atrial myocytes (pAMs) on days 3 and 9 of differentiation.

[0025] Figure 3 This image shows the results of detecting changes in corresponding electrophysiological parameters by optical voltage (or calcium) mapping after treating iAM-1 cells on day 9 of differentiation with specific ion channel agonists or blockers (sodium, potassium, and calcium channels). Figure 3 In the figure, A represents the action potential light signal trajectory results under different concentrations of TTX (0.3 μM, 1 μM) treatment at 1 Hz electrical stimulation; Figure 3 In the figure, B represents the representative action potential light signal trajectory results of the 0.3 μM TTX treatment group and the control group under 1 Hz electrical stimulation; Figure 3 In the figure, C represents the statistical comparison of conduction velocity (CV) between the 0.3 μM and 1 μM TTX treatment groups and the control group; Figure 3 In the figure, D represents the action potential light signal trajectory result under 1 Hz electrical stimulation under 100 nM astemizole treatment; Figure 3 In the figure, E represents the representative action potential light signal trajectory results of the 100 nM astemizole treatment group and the control group under 1 Hz electrical stimulation; Figure 3In the figure, F represents the statistical comparison results of APD40 and APD90 between the 100 nM astemizole treatment group and the control group; Figure 3 In the figure, G represents the calcium transient light signal trajectory result under 1 Hz electrical stimulation after treatment with 50 nM BayK8644 and 3 μM nifedipine. Figure 3 In the figure, H represents the representative calcium transient light signal trajectory results of the 50 nM BayK8644 (L-type calcium channel agonist) treatment group compared with the control group under 1 Hz electrical stimulation. Figure 3 The "I" in the figure represents the statistical comparison of calcium transient amplitude (CaT) between the 50 nM BayK8644 treatment group and the control group. Figure 3 In the figure, J represents the calcium transient light signal trajectory results under different frequencies (1, 2, 4, 6, 8, 10 Hz) of electrical stimulation under 50 nM BayK8644 treatment; Figure 3 In the figure, K represents the statistical comparison results of calcium transient time history (CaD80) at different frequencies between the 50 nM BayK8644 treatment group and the control group. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0027] The following description is based on specific embodiments.

[0028] Example 1: A method for screening drugs with cardiac ion channel toxicity In this embodiment, a conditional atrial myocardial cell (iAM) line was cultivated by introducing a lentiviral system expressing SV40 large T antigen (SV40LT antigen) induced by the tet-on system into cardiomyocytes. iAMs proliferated rapidly under doxycycline (dox) induction. After doxycycline (dox) removal, iAMs differentiated into cells similar in structure and functional maturity to primary cardiomyocytes. The differentiated iAM-1 monolayer cells exhibited more uniform and faster action potential (AP) conduction, similar to primary cells. It is proposed to use the atrial myocardial cell line (iAM-1) to measure the action potentials and conduction durations of rhythmic cardiomyocytes in 2D cell layers within the physiological heart rate range of cardiomyocytes using optical mapping techniques to identify the electrophysiological effects of drugs throughout the physiological heart. This method enables artifact-free and synchronized electrical stimulation of cardiomyocytes, allowing for average and objective analysis of AP. The iAM-1 cells used in this embodiment, as well as their basic culture, differentiation, and methods, were all implemented according to the methods published in the literature Liu J, Volkers L, Jangsangthong W, et al. Generation and primary characterization of iAM-1, a versatile new line of conditionally immortalized atrial myocytes with preserved cardiomyogenic differentiation capacity. Cardiovascular Research, 2018, 114(14): 1848–1859. The iAM-1 cell construction included the following steps: 1. Lentiviral vector construction: A lentiviral vector containing SV40 large T antigen (SV40 LT antigen) was constructed to induce its expression in cardiomyocytes.

[0029] (1) Obtaining the SV40 LT antigen gene: Using plasmid pLV[Exp]-Neo-EF1A>SV40-T (Yunzhou Biotechnology (Guangzhou) Co., Ltd.) as a template, a tsA58 mutation (A438V) was introduced through site-directed mutagenesis to obtain the temperature-sensitive SV40 LT antigen coding sequence (tsA58-LT). Using pLV[Exp]-Neo-EF1A>SV40-T as a template, PCR amplification was performed using specific primers (as shown in Table 1) to obtain an approximately 2.5 kb LT coding sequence. The PCR product was inserted into the pJET1.2 / blunt vector to construct the pJet1.2.LT-tsA58 plasmid, and the sequence was confirmed to be correct by sequencing.

[0030] Table 1 Note: The enzyme used for PCR amplification was VELOCITY DNA polymerase (GC Biotech, Alphen aan den Rijn, the Netherlands, catalog number: 60100). The PCR system (50 μL) consisted of: 25 μL of 2×Velocity PCR premix, 1 μL each of forward and reverse primers (10 μmol / L), 2 μL of template DNA (50 ng / μL), and sterile deionized water to a final volume of 50 μL. Amplification conditions: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 15 s, 58℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 35 cycles, with a final extension at 72℃ for 10 min, and storage at 4℃.

[0031] (2) Selection and modification of lentiviral vector backbone: To achieve specific and inducible expression of the target gene in cardiomyocytes, this embodiment selected the Tet-on inducible lentiviral shuttle vector pLVET-tTR-KRAB (purchased from Addgene, catalog number: 11644) as the starting backbone. This original vector contains a human extension factor 1α (EF1α) promoter, an enhanced green fluorescent protein (eGFP) coding sequence, and a Tet-on inducible regulatory element (tTR-KRAB), which can induce the expression of the target fragment under doxycycline (dox) regulation. However, the EF1α promoter is a pan-cellular promoter and cannot achieve cardiomyocyte-specific expression; therefore, it needs to be specifically modified.

[0032] The core objective of the modification is to replace the EF1α promoter-eGFP expression cassette in the original vector with a skeletal muscle-specific MHCK7 promoter and multiple cloning site (MCS). This allows the modified vector to efficiently express the downstream target gene exclusively in cardiomyocytes under dox induction via the MHCK7 promoter, eliminating non-specific expression in non-cardiomyocyte cells. The specific modification steps are as follows: 1) Vector digestion and large fragment recovery: The pLVET-tTR-KRAB vector was double-digested with restriction endonucleases SpeI and EcoRI (both purchased from Thermo Fisher Scientific, catalog numbers EN0321 and EN0271, respectively) (digestion system: 5 μL 10× digestion buffer, 1 μL SpeI (10 U / μL), 1 μL EcoRI (10 U / μL), 10 μg vector plasmid, and sterile deionized water to a final volume of 50 μL; incubated at 37°C for 3 h). The digestion products were separated by 1% agarose gel electrophoresis (120 V, 30 min). The large fragment of the vector (approximately 11.0 kb) was recovered using a gel recovery kit (purchased from Qiagen, catalog number 12143) and stored at -20°C for later use.

[0033] 2) PCR amplification of the MHCK7 promoter: Specific primers were designed based on the MHCK7 promoter sequence in GenBank (accession number: NM_001001460.1). The upstream primer had a SpeI restriction site at its 5′ end (sequence shown in Table 2), and the downstream primer had an EcoRI restriction site at its 5′ end (sequence shown in Table 2). (Primers were supplied by Eurofins MWG Operon, Ebersberg). (Synthesized in Germany); Genomic DNA from neonatal rat myocardial tissue (extracted from the atrial tissue of 2-day-old Wistar rats, using the QIAGEN Genomic DNA Extraction Kit, catalog number 69104) was used as a template for PCR amplification (PCR system: 25 μL of 2×Taq enzyme premix, 1 μL each of forward and reverse primers (10 μmol / L), 2 μL of template DNA (50 ng / μL), and sterile deionized water to 50 μL; amplification conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 35 cycles, with a final extension at 72℃ for 10 min); The amplified product was verified by 1% agarose gel electrophoresis (target fragment size approximately 1.2 kb), and after gel recovery, the purified MHCK7 promoter fragment was obtained and stored at -20℃ for later use.

[0034] 3) Multiple Cloning Site Adapter Ligation and Vector Construction: Multiple cloning site (MCS) adapters containing commonly used restriction enzyme sites such as EcoRI, BamHI, and XhoI were artificially synthesized (sequences shown in Table 2, synthesized by Eurofins MWG Operon). The MHCK7 promoter fragment and the MCS adapter were ligated using T4 DNA ligase (Thermo Fisher Scientific, catalog number EL0011) (ligation system: 2 μL 10× ligation buffer, 5 μL MHCK7 fragment (50 ng / μL), 2 μL MCS adapter (100 ng / μL), 1 μL T4 DNA ligase (5 U / μL), sterile deionized water to a final volume of 20 μL, ligated overnight at 16°C). The ligation product (MHCK7-MCS fragment) was then ligated with the large vector fragment recovered in step 1 (ligation system: 5 μL 10× ligation buffer, 8 μL large vector fragment (50 ng / μL), 5 μL MHCK7-MCS ligation product, T4…). Add 2 μL of DNA ligase and sterile deionized water to a final volume of 50 μL, and ligate overnight at 16°C to obtain the recombinant vector ligation solution.

[0035] Table 2 4) Positive clone screening and sequence verification: The recombinant vector ligation solution was transformed into *E. coli* DH5α competent cells (purchased from Thermo Fisher Scientific, catalog number 18265017). After incubating on ice for 30 min, the cells were heat-shocked at 42°C for 90 s, immediately followed by an ice incubation for 2 min. 500 μL of antibiotic-free LB broth was added, and the cells were incubated at 37°C with shaking at 200 rpm for 1 h. 100 μL of the culture was then spread onto LB solid medium containing ampicillin (final concentration 100 μg / mL) and incubated upside down at 37°C for 12–16 h. Single colonies were picked and inoculated into LB broth containing ampicillin (final concentration 100 μg / mL) and incubated overnight at 37°C with shaking at 200 rpm. Ampicillin resistance screening, based on the *E. coli* β-lactamase gene (Amp) contained in the original vector, can rapidly screen for positive colonies containing the recombinant vector.

[0036] 5) Recombinant Vector Identification: Plasmid DNA was extracted from single colonies (plasmid extraction kit purchased from Qiagen, catalog number 12143), and subjected to SpeI and EcoRI double enzyme digestion and PCR identification. The double enzyme digestion system was the same as in step 1. After digestion, a vector fragment of approximately 11.0 kb and an MHCK7-MCS fragment of approximately 1.3 kb should appear. For PCR identification, the recombinant plasmid was used as a template, and MHCK7 promoter-specific primers were used. The amplification conditions were the same as in step 2, and a target band of approximately 1.2 kb should be obtained. Clones that were positive for both enzyme digestion and PCR identification were sent to Invitrogen for sequencing verification to confirm that the MHCK7 promoter sequence, MCS sequence, and vector ligation site were correct and without base deletions, mutations, or reverse ligation.

[0037] After the above modifications, a Tet-on-inducible, cardiomyocyte-specific lentiviral shuttle vector, named pLV.iMHCK7, was obtained. This vector retains the Tet-on-inducible regulatory element (tTR-KRAB) of the original vector. In the presence of dox (final concentration 1 μg / mL), it can efficiently drive the downstream target gene to be specifically expressed in cardiomyocytes through the skeletal muscle-specific MHCK7 promoter, while showing almost no expression in non-cardiomyocytes (such as hepatocytes and fibroblasts). This effectively solves the off-target problem caused by the generalized expression of the original vector, laying the foundation for subsequent targeted regulation research and applications of the target gene in cardiomyocytes.

[0038] (3) Construction of recombinant lentiviral vector The SV40 LT antigen gene fragment (tsA58-LT, approximately 2.5 kb) obtained in step (1) was inserted into the MssⅠ and EcoRI sites of the lentiviral shuttle vector pLV.iMHCK7 to construct the recombinant lentiviral shuttle plasmid pLV.iMHCK7.LT-tsA58. Specific operation: Take pJet1.2.LT-tsA58 plasmid and pLV.iMHCK7 vector, and perform double digestion with MssI (Thermo Fisher Scientific, catalog number EN0541) and EcoRI (Thermo Fisher Scientific, catalog number EN0271) respectively. The digestion system is the same as the vector digestion in step (2). Incubate at 37℃ for 3h. After the digestion product is recovered by gel, the tsA58-LT gene fragment (about 2.5kb) and the pLV.iMHCK7 vector large fragment (about 11.0kb) are obtained. The two are ligated by T4 DNA ligase (Thermo Fisher Scientific, catalog number EL0011) (ligation system: 5μL of 10× ligation buffer, 8μL of vector large fragment, 5μL of tsA58-LT gene fragment, 2μL of T4 DNA ligase, and sterile deionized water to make up to 50μL. Ligate overnight at 16℃) to obtain the recombinant vector ligation solution. The recombinant vector ligation solution was transformed into Escherichia coli DH5α competent cells, and the cells were screened on LB plates containing 100 μg / mL ampicillin. Positive clones were selected for PCR and enzyme digestion identification.

[0039] (4) Screening and identification of recombinant vectors Plasmids were extracted from positive clones (using the Qiagen Plasmid Extraction Kit, catalog number 12143). Double digestion with restriction endonucleases MssI and EcoRI was performed for identification. The digestion system consisted of 5 μL 10× digestion buffer, 1 μL MssI (10 U / μL), 1 μL EcoRI (10 U / μL), 10 μg recombinant plasmid, and sterile deionized water to a final volume of 50 μL. The mixture was incubated at 37°C for 3 h. The digestion products were analyzed by 1% agarose gel electrophoresis, which showed approximately 2.5 kb of the SV40 LT antigen gene insert and approximately 11.0 kb of the vector fragment. Further sequencing (Invitrogen) confirmed that the insert sequence was completely identical to the tsA58-LT coding sequence, with no base mutations, deletions, or reverse insertions. Positive clones with correct sequences were retained, expanded, and plasmids were extracted and stored at -20°C for subsequent experiments.

[0040] (5) Packaging and concentration of lentiviruses The recombinant lentiviral shuttle plasmid pLV.iMHCK7.LT-tsA58 was co-transfected into 293T cells with helper packaging plasmids psPAX2 (Addgene, catalog number: 12260) and pLP / VSVG (Thermo Fisher Scientific, catalog number: K497500) at a molar ratio of 2:1:1 (confluence approximately 70-80%). The 293T cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM; Life Technologies Europe, catalog number: 41966) containing 10% fetal bovine serum (FBS; Life Technologies Europe, catalog number: 10270106) at 37°C, 5% CO2, and saturated humidity.

[0041] Transfection was performed using the polyethyleneimine (PEI) method. Specific procedures included: per 175-cm²... 2 For a cell culture flask (Greiner Bio-One, catalog number 660160), take 35 μg of plasmid DNA (pLV.iMHCK7.LT-tsA58 17.5 μg, psPAX2 8.75 μg, pLP / VSVG 8.75 μg) and 105 μg of PEI (Polysciences Europe, catalog number 23966), dissolve them in 2 ml of 150 mM NaCl solution, mix gently, and incubate at room temperature for 15 min to form a DNA-PEI complex. Add the complex directly to the 293T cell culture medium and gently shake the culture flask to distribute the complex evenly.

[0042] The morning after transfection, the transfection medium was replaced with 15 ml of fresh high-glucose DMEM supplemented with 5% FBS and 25 mM HEPES-NaOH (pH 7.4, Sigma-Aldrich, catalog number H3375). Approximately 48 hours after the start of transfection, the cell culture supernatant was collected, centrifuged at 3750×g at room temperature for 10 minutes to remove cell debris, and then filtered through a polyethersulfone Millex-HP syringe filter (Millipore, catalog number SLHV033RB) with a pore size of 0.45 μm and a diameter of 33 mm to further remove impurities.

[0043] The filtered viral supernatant was placed in a 38.5 ml polypropylene ultracentrifuge tube (Beckman Coulter Nederland, catalog number 355622). A 5 ml layer of 20% (w / v) sucrose phosphate-buffered saline (PBS) solution (sucrose purchased from Sigma-Aldrich, catalog number S0389; PBS purchased from Thermo Fisher Scientific, catalog number 10010023) was added to the bottom layer. The tube was centrifuged at 15,000 rpm for 120 minutes at 4°C using an SW32 rotor (Beckman Coulter Nederland) with slow acceleration and no brakes. After centrifugation, the supernatant was carefully discarded, and the viral pellet at the bottom of the tube was resuspended in 1 ml PBS with 1% bovine serum albumin (BSA; Sigma-Aldrich, catalog number A7906). The suspension was gently shaken overnight at 4°C to dissolve the virus, obtaining a concentrated lentivirus suspension. The concentrated viral suspension was aliquoted into 50 μL portions on ice and stored at -80°C for later use, avoiding repeated freeze-thaw cycles.

[0044] (6) Lentiviral titer determination Lentiviral titers were determined using a limiting dilution method. 293T cells were seeded into 96-well plates, and serially diluted viral solutions were added. After incubation for 48-72 hours, the integrated viral copy number was detected using qPCR, and the viral titer (TU / mL) was calculated. Wells with viral copy numbers between 10-100 were selected, and the viral titer (TU / mL) was calculated using the following formula: Viral titer = (Viral copy number × Cell number × Dilution factor) / Viral solution volume (mL). The average of the results from three replicates for each dilution gradient was taken as the final viral titer, which should be ≥1×10⁻⁶. 8 Only when the concentration of TU / mL is reached can it be used for subsequent cardiomyocyte transfection experiments.

[0045] 2. Cell infection: The constructed lentiviral vector is used to infect cardiomyocytes. After a period of culture, cells that express the vector stably are selected to obtain the dox-induced atrial myocyte (iAMs) line.

[0046] (1) Preparation of cardiomyocytes: Newborn Wistar rats (within 2 days of birth) were anesthetized with 4%-5% isoflurane inhalation. After confirming the absence of pain reflexes, the hearts were removed under aseptic conditions. Atrial tissue was separated from the ventricles, minced, and digested twice for 30 minutes each time using type I collagenase (Worthington Biochemical, catalog number: LS004196) and DNase I (Sigma-Aldrich, catalog number: DN25). Cells were collected by centrifugation at 160×g for 10 minutes and resuspended in Ham's F10 medium containing 10% heat-inactivated FBS and 10% heat-inactivated horse serum. The cell suspension was transferred to Primaria culture dishes and incubated at 37°C and 5% CO2 for 120 minutes to remove adherent non-cardiac cells (mainly fibroblasts). Non-adherent cells (mainly atrial myocytes) were collected, filtered through a 70 μm cell sieve, and then digested at 2×10⁻⁶ cells per cell line. 3 cells / cm 2 The inoculum was seeded at a density in culture dishes coated with bovine plasma fibronectin.

[0047] (2) Lentiviral infection: Primary atrial myocytes (pAMs) were infected with lentivirus on day 1 after inoculation, with the virus added at an MOI of 10. The medium was replaced the next day with 100 ng / mL doxycycline (dox) to induce LT antigen expression. Cell lysates were collected after 7 days of culture for Western blot analysis. Mouse anti-SV40 large T antigen monoclonal antibody (Santa Cruz Biotechnology, catalog number: sc-147, dilution 1:2000) was used as the primary antibody, and GAPDH was used as the internal control. The results are as follows: Figure 1 As shown, a clear SV40 large T antigen protein band was detected in the induction group, while it was not detected in the control group (without dox), indicating that the constructed lentiviral vector can induce the expression of SV40 large T antigen in cardiomyocytes.

[0048] (3) Cultivation and screening: Following viral infection, cells were cultured in medium containing 100 ng / mL dox. The medium was changed every 2-3 days. After one week of culture, cells were digested with trypsin at a concentration of 10-20 cells / cm³. 2 Low-density seeding was performed in 100 mm cell culture dishes, and single-cell colonies were formed after 2-3 weeks of culture. When the colonies reached a visible size, single cell colonies were picked using a cloning loop and transferred to new culture dishes for amplification in Dox-containing medium. The amplified clones were subjected to induction differentiation ability tests, and clones with better electrophysiological performance after differentiation (such as the clone in this study, named iAM-1) were selected to obtain the tet-on inducible atrial myocardial cell line. All screening processes were carried out in a 37°C, 5% CO2 incubator.

[0049] 3. Cell proliferation and differentiation: Doxycycline (Dox) was added to the culture medium to induce rapid proliferation of iAMs cells. Once the cells reached a suitable density, doxycycline (Dox) was removed, allowing the iAMs cells to differentiate into cells with structure and function similar to primary cardiomyocytes.

[0050] (1) Induction of iAMs cell proliferation: iAMs cells were digested with trypsin and then incubated at a concentration of 8000 cells / cm². 2 The cells were seeded at a density of [insert density here] in culture dishes, and doxycycline (dox, Sigma-Aldrich) was added to the culture medium to a final concentration of 100 ng / mL. The dishes were then incubated in a 37°C, 5% CO2 cell culture incubator, with the medium replaced with fresh dox-containing medium every 2-3 days. Under these conditions, the average doubling time of iAMs was approximately 38 hours.

[0051] (2) Cell differentiation induction: iAMs cells were digested with trypsin and seeded at an appropriate density in culture plates pre-coated with bovine plasma fibronectin (Sigma-Aldrich, catalog number: F1141) to remove dox and differentiate into cells with similar structure and function to primary cardiomyocytes.

[0052] (3) Differentiation identification: Electrophysiological function verification: Monolayer cells were detected by optical voltage mapping (8 × 10⁶ cells per well in a 24-well plate). 5 Action potential duration and conduction velocity (APD) of individual atrial myocytes (iAMs) under 1 Hz electrical stimulation. The APD and CV of iAMs on day 9 of differentiation should not differ significantly from those of primary atrial myocytes.

[0053] 4. Drug screening experiments (1) Cell seeding: iAM-1 cells were seeded at a rate of 8 × 10⁶ cells / year. 5 Cells / wells were seeded at a density of 12 mm on round glass coverslips pre-coated with bovine plasma fibronectin in 24-well plates, and cultured and differentiated for 9 days after removing dox, for use in optical mapping experiments.

[0054] (2) Optical labeling: Cells were labeled using voltage-sensitive dye Di-4-ANEPPS (manufacturer: Thermo Fisher Scientific (Invitrogen); catalog number: D1199) or calcium ion-sensitive dye Rhod-2-AM (manufacturer: Thermo Fisher (Invitrogen); catalog number: R1244) according to the dye instructions.

[0055] (3) Drug treatment: The drugs to be tested (Nav1.5 blocker tetrodotoxin TTX citrate Almone T-550, hERG blocker astemizole, Cav1.2 agonist BayK8644 or Cav1.2 blocker nifedipine) were dissolved in appropriate solvents (TTX citrate dissolved in pure water, others dissolved in DMSO) to prepare drug solutions with a final concentration of 1000×, namely 1mM, 100μM, 50μM, and 3mM. The drug solutions were added to the culture dishes containing iAM-1 cells, and a blank control group without drug was set up (an equal volume of solvent was added, control). Each group had more than 3 replicates. The drug treatment time was 20 minutes.

[0056] (4) Optical voltage mapping: The culture dish was placed in an optical mapping system (manufacturer: SciMedia; model: MiCAMULTIMA-L). The mapping system is equipped with a 100×100 pixel CMOS sensor (sensor size 10 mm×10 mm), with a maximum frame rate of 10,000 fps, which can accurately capture the rapid propagation of action potentials and calcium transients in a cardiomyocyte monolayer. Local single-point electrical stimulation was performed on the cell monolayer using epoxy resin-coated platinum electrodes. The stimulation parameters were set as follows: pulse width 2 ms, voltage amplitude 8 V, frequency 1-10 Hz, controlled using an STG 2004 stimulator (Multi Channel Systems) and its accompanying software. Excessive stimulation was avoided to prevent cell damage. The ambient temperature was kept constant at 37℃ during the mapping process to avoid vibration interference.

[0057] 5. Data Analysis: The recorded data was processed and analyzed using Brain Vision Analyzer 1208 software (manufacturer: BrainVision, Japan). The analysis included: Propagation velocity (CV): The propagation velocity of the excitation wavefront is determined by calculating the isochronous spacing using the activation map.

[0058] Action potential duration (APD): APD values ​​were measured at 40% and 90% repolarization.

[0059] Calcium transient analysis (for calcium-sensitive dye experiments): Analyzes the amplitude and duration of calcium transients.

[0060] Statistical analysis: Electrophysiological parameters of different drug concentration groups and control groups were compared. Nested ANOVA combined with Bonferroni post-hoc test was used for statistical analysis. P < 0.05 was considered statistically significant.

[0061] For specific analysis methods, please refer to the data analysis workflow described in the SciMedia official documentation. Optical mapping technology can achieve high-throughput, high spatiotemporal resolution recording of membrane potential and calcium signals, and has significant advantages over traditional electrophysiological techniques such as patch clamp, including non-destructive and simultaneous detection of multiple signals.

[0062] The above method can quickly and accurately determine the type and intensity of the toxic effects of the drug on cardiac ion channels, and is suitable for high-throughput screening of cardiotoxic drugs.

[0063] 6. Results The measurement results are as follows Figure 2 As shown in Figures A through D, the conditional cardiomyocyte line (iAM) exhibits a gradual maturation of its electrophysiological function during the withdrawal of doxycycline (dox)-induced differentiation. Figure 1 As shown in A~D, the action potential morphology at five key time points (days 0, 1, 2, 3, and 9) of differentiation was examined by optical mapping. It can be seen that in the early stage of differentiation (days 0-2), the cells have no obvious action potential (no signal); by day 3 of differentiation, the action potential begins to show obvious action potential; by day 9 of differentiation, the action potential morphology is highly consistent with that of primary atrial myocytes (pAMs). Figure 1 Statistical analysis of B further showed that compared with day 3 of differentiation, the action potential durations (APD40 and APD90) of iAM cells on day 9 of differentiation were significantly shortened (P<0.01), while the conduction velocity (CV) was significantly accelerated (P<0.001); and the APD and CV values ​​at this time point were not statistically different from those of primary atrial myocardial cells (pAMs) cultured for 9 days (P>0.05). These results demonstrate that iAM cells on day 9 of differentiation have reached a level of electrophysiological maturity comparable to primary cells, providing a stable and reliable cell model for this drug screening method.

[0064] like Figure 2 As shown, iAM-1 cells on day 9 of differentiation were treated with specific ion channel modulators. Optical mapping clearly detected the expected changes in cardiac electrophysiological parameters, thus verifying the specificity of the cell line's response to different ion channel modulators and the screening capability of this method. Specifically: sodium channel blocking verification ( Figure 3 (As shown in A~C): Cells treated with different concentrations of tetrodotoxin (TTX, a Nav1.5 specific blocker) showed a dose-dependent decrease in conduction velocity of cell monolayers under 1 Hz electrical stimulation. 0.3 μM TTX reduced conduction velocity by approximately 40%, while 1 μM TTX resulted in a decrease of over 80% (P<0.001), indicating that this method can sensitively detect the inhibitory effect of sodium channel blockers on myocardial electrical conduction.

[0065] Potassium channel blocking verification ( Figure 3 (As shown in D~F): Cells treated with 100 nM astemizole (a specific blocker of hERG / Kv11.1) showed a significant prolongation of action potential duration (APD) upon 1 Hz electrical stimulation. Specifically, APD90 increased from approximately 120 ms in the control group to approximately 220 ms (P<0.01), mimicking the drug-induced long QT syndrome phenotype, demonstrating that this method can effectively assess the risk of hERG channel-related cardiotoxicity.

[0066] Calcium channel regulation verification ( Figure 3 As shown in G~I): Treatment of cells with the L-type calcium channel agonist BayK8644 (50 nM) increased the calcium transient (CaT) amplitude by approximately 2.5 times compared to the control group (P<0.01); while treatment with the L-type calcium channel blocker nifedipine (3 μM) reduced the calcium transient amplitude to approximately 30% of the control group (P<0.01). These contrasting changes suggest that this method can distinguish between calcium channel activation and blockade.

[0067] Frequency dependency verification ( Figure 3 (J~K in the diagram): The effect of BayK8644 on calcium transient duration (CaD) was observed at different stimulation frequencies (1, 2, 4, 6, 8, 10 Hz). The results showed that BayK8644 significantly prolonged CaD in the heart rate range of 4 Hz and below, while the effect weakened at higher frequencies. This indicates that this method can assess the frequency-dependent effect of drugs under different heart rate conditions, making up for the shortcomings of traditional patch-clamp and MEA techniques in detecting within the heart rate range.

[0068] In summary, the conditional cardiomyocyte cell line (iAM) established in this invention exhibits electrophysiological functions highly consistent with primary atrial myocytes after day 9 of differentiation. By combining this cell line with optical (voltage / calcium) mapping technology, and detecting multiple electrophysiological indicators such as conduction velocity, action potential duration, and calcium transient amplitude and duration, the agonist or antagonist effects of drugs on cardiac sodium, potassium, and calcium ion channels can be sensitively and specifically reflected. Therefore, this method is simple to operate, provides objective results, and allows for parallel detection of multiple indicators, making it suitable for high-throughput screening of cardiotoxic drugs. It has significant advantages over existing technologies (such as heterologous expression systems, patch-clamp, and MEA).

[0069] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0070] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for screening drugs with cardiac ion channel toxicity, characterized in that, include: Conditional cardiomyocyte cell lines were cultured by infecting cardiomyocytes with a lentiviral system carrying the SV40 large T antigen. The conditional cardiomyocyte lines were labeled using voltage-sensitive dyes or calcium ion-sensitive dyes. The drug to be tested and the buffer solution were added to a culture dish containing labeled conditional cardiomyocytes to form a test group and a control group. Optical voltage calibration was used to test the test group and the control group, and statistical significance analysis was performed on the optical voltage calibration results. In the optical mapping, the electrical stimulation method is point stimulation; the stimulation parameters of the electrical stimulation are: pulse width 1~3ms, voltage amplitude 6~9V, and frequency 1-10 Hz.

2. The method as described in claim 1, characterized in that, The test drug includes at least one of the following: a voltage-gated sodium channel Nav1.5 blocker, a voltage-gated potassium channel Kv11.1 / hERG blocker, an L-type calcium channel Cav1.2 agonist, and an L-type calcium channel Cav1.2 blocker.

3. The method as described in claim 2, characterized in that, The voltage-gated sodium channel Nav1.5 blocker includes tetrodotoxin; the voltage-gated potassium channel Kv11.1 / hERG blocker includes astemizole; the L-type calcium channel Cav1.2 agonist includes BayK8644; and the L-type calcium channel Cav1.2 blocker includes nifedipine.

4. The method as described in claim 1, characterized in that, The optical voltage detection results include: action potential amplitude, action potential duration, conduction velocity, calcium transient amplitude, and calcium transient duration for the test group and the control group.

5. The method as described in claim 1, characterized in that, The SV40 large T antigen is a temperature-sensitive mutant tsA58.

6. The method as described in claim 1, characterized in that, The lentiviral system employs the Tet-on / tTS tetracycline-induced expression system to achieve doxycycline-dependent expression of the large T antigen.

7. The method as described in claim 1, characterized in that, The expression of the large SV40 large T antigen is driven by a myocardial-specific promoter.

8. The method as described in claim 7, characterized in that, The myocardial-specific promoter is the MHCK7 promoter.

9. The method as described in claim 1, characterized in that, Cardiomyocytes were infected using a lentiviral system carrying the SV40 large T antigen in a medium containing doxycycline.

10. The method as described in claim 1, characterized in that, The multiplicity of infection (MOI) for cardiomyocytes infected with a lentiviral system carrying the SV40 large T antigen ranges from 1 to 10.

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