Application of calcium ion indicator GCaMP in calcium signal detection of human myocardial cell line

Abstract

The invention provides application of a calcium ion indicator GCaMP in detection of calcium signals of a human myocardial cell line, the calcium ion indicator GCaMP is successfully applied to detection of calcium signals of myocardial cells, and the application of the calcium ion indicator GCaMP in detection of calcium signals of myocardial cells is increased. Secondly, the invention provides a method for detecting cardiac muscle cell calcium signals based on a calcium ion indicator GCaMP and detecting the excitation and / or inhibition effect of a small molecule compound on a cardiac muscle cell PIEZO1 channel, and a foundation is laid for researching cardiac muscle cell related calcium channels.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to application of a calcium ion indicator GCaMP in calcium signal detection of a human myocardial cell line. Background Art

[0002] Calcium is the core element of the excitation-contraction coupling of cardiomyocytes. Under external stimulation, calcium ions enter the cell from the outside through calcium channels, increasing the local calcium ion concentration and activating the sarcoplasmic reticulum Ryanodine Receptor (Ryr), which ultimately causes myofilament sliding and promotes cardiomyocyte contraction through calcium-induced calcium release. During the myocardial diastole, some calcium ions are taken back into the sarcoplasmic reticulum for the next contraction. As a second messenger, calcium is widely involved in various signal transductions that cardiomyocytes respond to. Cardiomyocytes use intracellular calcium ions as a medium to respond to external environmental changes and initiate various downstream physiological or pathological reactions to cope with environmental changes. For many years, calcium signals and their related research on the development of cardiomyocytes and their physiological and pathological processes have been the focus of research in this field.

[0003] The PIEZO1 channel is a new type of cardiac mechanical stress sensor. Under normal circumstances, the PIEZO1 channel is closed, but under the stimulation of mechanical stretch, PIEZO1 channel agonist Yoda1, etc., the PIEZO1 channel opens, increases calcium influx, destroys intracellular calcium ion homeostasis, and then affects downstream signaling pathways to promote the occurrence and development of diseases. Therefore, in-depth research on calcium signals in cardiomyocytes can promote the research progress of PIEZO1 channels and a series of calcium-related channels.

[0004] Calcium imaging refers to the method of using calcium ion indicators to monitor calcium ion concentration in tissues. Among calcium ion indicators, genetically encoded fluorescent calcium ion indicators can be divided into two categories: single-fluorophore genetically encoded calcium indicators and FRET-based / two-fluorescent protein genetically encoded calcium indicators. Among them, the single-fluorophore genetically encoded GCaMP family is the most widely used. GCaMP is composed of circularly permuted Enhanced Green Fluorescent Protein (cpEGFP), calmodulin (CaM) and a polypeptide sequence (M13) of myosin light chain kinase. In the absence of calcium ions, cpEGFP cannot function; when CaM-M13 specifically binds to calcium ions, cpEGFP undergoes conformational changes and is excited to release green fluorescence.

[0005] At present, the detection of myocardial cell calcium signals is still mainly based on chemical fluorescent dyes, but due to the inaccurate subcellular localization, the long-term incubation of chemical dyes will cause toxicity to cells, and the short-term residence of dyes in cells limits the observation time, the research progress of cardiac myocardial cells is limited. Therefore, the development of a method that can better detect myocardial cell calcium signals is very necessary for the study of myocardial cell calcium signals. Summary of the invention

[0006] The purpose of the present invention is to provide an application of a calcium ion indicator GCaMP in the detection of calcium signals in human myocardial cell lines.

[0007] The present invention adopts the following technical solution:

[0008] Application of a calcium ion indicator GCaMP in the detection of calcium signals in human cardiomyocyte cell lines.

[0009] Application of lentivirus packaged with calcium ion indicator GCaMP in calcium signal detection of human cardiomyocyte cell line.

[0010] A method for detecting calcium signals in human cardiomyocyte cell lines using lentivirus packaged with the calcium ion indicator GCaMP.

[0011] Furthermore, the method for detecting calcium signals in human myocardial cell lines using a lentivirus packaged with a calcium ion indicator GCaMP comprises the following steps:

[0012] (1) Recovery, culture, digestion and subculture of human cardiomyocytes;

[0013] (2) lentiviral infection of human cardiomyocyte cell lines;

[0014] (3) 48 h after infection, the cells were observed under a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed and statistically analyzed.

[0015] Wherein, step (2) specifically includes:

[0016] a) Plating the human cardiomyocytes treated in step (1) into a well plate;

[0017] b) Calculate the amount of lentivirus added to each well according to the most suitable multiplicity of infection, and prepare the infection solution with DMEM high-glucose medium containing 5 μg / mL Polybrene;

[0018] c) Discard the old culture medium in the well plate and add the infection solution; after 16 hours of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-glucose culture medium, and continue to culture until infection for 48 hours.

[0019] Preferably, the optimal MOI in step (2) is 20.

[0020] Furthermore, the lentivirus is pSLenti-CMV-jGCaMP7f-PGK-Puro-WPRE.

[0021] The invention discloses an application of a lentivirus packaged with a calcium ion indicator GCaMP in detecting the agonist and / or inhibitory effect of a compound on the PIEZO1 channel of a human cardiomyocyte cell line.

[0022] A method for detecting the agonist and / or inhibitory effect of a compound on the PIEZO1 channel of a human myocardial cell line, comprising the following steps:

[0023] (1) Recovery, culture, digestion and subculture of human cardiomyocyte cell line AC16;

[0024] (2) lentivirus infection of human cardiomyocyte cell line AC16 treated in step (1);

[0025] I) Plating the human cardiomyocytes AC16 treated in step (1) into a well plate;

[0026] II) Divide the plate into two groups and perform lentiviral infection separately:

[0027] (i) Control group A: Calculate the amount of lentivirus per well according to MOI = 20, and prepare the infection solution with DMEM high-glucose medium containing 5 μg / mL Polybrene; discard the old medium in the well plate and add the infection solution; after 16 hours of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-glucose medium, and culture until infection for 48 hours; continue to culture for 24 hours;

[0028] (ii) Experimental group A: The amount of lentivirus added to each well was calculated according to MOI = 20, and the infection solution was prepared with DMEM high-glucose medium containing 5 μg / mL Polybrene; the old medium in the well plate was discarded and the infection solution was added; after 16 hours of infection, the infection solution was discarded, PBS was washed twice, and fresh DMEM high-glucose medium was added, and cultured until infection for 48 hours; 20 μM of the small molecule compound to be tested was added and cultured for another 24 hours;

[0029] (3) Observe under a fluorescence microscope, perform quantitative analysis on the fluorescence intensity, and collect statistical data; obtain the relative fluorescence intensity of the experimental group A relative to the control group A, and the small molecule compound to be tested that increases by more than 20% compared with the control group is an agonist of the PIEZO1 channel;

[0030] (4) selecting small molecule compounds to be tested whose relative fluorescence intensity of experimental group A is not significantly different from that of control group A;

[0031] (5) lentivirus infection of human cardiomyocyte cell line AC16 treated in step (1);

[0032] I) Plating the human cardiomyocytes AC16 treated in step (1) into a well plate;

[0033] II) Divide the plate into two groups and perform lentiviral infection separately:

[0034] (i) Control group B: Calculate the amount of lentivirus per well according to MOI=20, and prepare the infection solution with DMEM high-glucose medium containing 5 μg / mL Polybrene; discard the old medium in the well plate and add the infection solution; after 16 hours of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-glucose medium, and culture until infection for 48 hours; add 20 μM Yoda1 and continue to culture for 24 hours;

[0035] (ii) Experimental group B: Calculate the amount of lentivirus to be added to each well according to MOI = 20, and prepare the infection solution with DMEM high-glucose medium containing 5 μg / mL Polybrene; discard the old medium in the well plate and add the infection solution; after 16 hours of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-glucose medium, and culture until infection for 48 hours; add 20 μM Yoda1 and 20 μM of the small molecule compound to be tested obtained by step (4) and continue to culture for 24 hours;

[0036] (6) Observe under a fluorescence microscope, perform quantitative analysis on the fluorescence intensity, and calculate the statistics; obtain the relative fluorescence intensity of the experimental group B relative to the control group B. The small molecule compound to be tested that decreases by more than 20% compared with the control group is an inhibitor of the PIEZO1 channel.

[0037] Furthermore, in step (4), a small molecule compound to be tested whose relative fluorescence intensity of the experimental group A relative to the control group A is between 80% and 120% is selected.

[0038] The beneficial effect of the present invention is that the present invention provides the application of the calcium ion indicator GCaMP in the detection of calcium signals in human cardiomyocytes, successfully applies the calcium ion indicator GCaMP to the detection of cardiomyocyte calcium signals, and increases the use of the calcium ion indicator GCaMP in the detection of cardiomyocyte calcium signals.

[0039] Secondly, the present invention provides a sample processing method for detecting myocardial cell calcium signals based on the calcium ion indicator GCaMP. Based on GCaMP, the stimulatory or inhibitory effects of small molecule compounds on the PIEZO1 channel of myocardial cells are detected, laying the foundation for studying myocardial cell-related calcium channels. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the verification of the infection efficiency in Example 1.

[0041] Figure 2 is the structural formula of compound MXY003-005.

[0042] Figure 3 This is the accuracy verification result of the method of the present invention.

[0043] Figure 4 This is a diagram showing the agonistic effect of the first group of compounds in Example 2.

[0044] Figure 5 This is a diagram showing the agonistic effect of the second group of compounds in Example 2.

[0045] Figure 6 This is a diagram showing the agonistic effect of the third group of compounds in Example 2.

[0046] Figure 7 This is a diagram showing the inhibitory effect of the first group of compounds in Example 3.

[0047] Figure 8 This is a diagram showing the inhibitory effect of the second group of compounds in Example 3.

[0048] Fig. 9 This is a diagram showing the inhibitory effect of the third group of compounds in Example 3. DETAILED DESCRIPTION

[0049] The present invention is described in detail below in conjunction with the embodiments and drawings.

[0050] Example 1 Detection of calcium signals in human cardiomyocyte cell line AC16

[0051] (1) Recovery, culture, digestion and subculture of human cardiomyocyte cell line AC16

[0052] Take the human cardiomyocyte cell line AC16 cells out of the -80℃ freezer and thaw them in a 37℃ water bath. When the cells are almost completely thawed, use a 1mL pipette to aspirate the cells in a clean bench, add them to 3mL of DMEM (high glucose) medium containing 10% FBS, and centrifuge at 1000rpm for 5min. Discard the supernatant, resuspend with 4mL of DMEM (high glucose) medium containing 10% FBS, and spread them in a 10cm culture dish. After shaking the cells, place them in a 37℃, 5% CO 2 , and cultured in a cell culture incubator with saturated humidity.

[0053] The composition of DMEM (high glucose) medium is as follows: 4.5 g / L D-glucose, 580 mg / L L-glutamine, and 110 mg / L sodium pyruvate (Gicbo, C11995500BT).

[0054] Observe the cell growth status every day, and change the cell medium every 1 to 2 days. Take cells with good growth status and a confluence rate of 90% for digestion and passage. Discard the culture medium, wash the cells twice with phosphate buffered saline (PBS), add 1mL of 0.25% trypsin + 0.02% EDTA solution preheated at 37°C to digest for 1 to 2 minutes, place the cells under a microscope for observation, and when more than half of the cells shrink and move in a sandy state, add 3mL of 10% FBS DMEM (high glucose) culture medium to terminate digestion. Place in a centrifuge and centrifuge at 1000rpm for 5 minutes. Discard the supernatant, add DMEM (high glucose) culture medium (90% DMEM (high glucose) culture medium + 10% FBS + 1% PS) to resuspend, and plate.

[0055] The specific components of PS are: Penicillin: 10000 U / mL, Streptomycin: 10000 μg / mL (VivaCell, C3420-0100). Pancreatic enzymes include trypsin, pancreatic amylase and pancreatic lipase (Senrui, CR25200).

[0056] (2) Determine the optimal multiplicity of infection (MOI) for lentiviral infection

[0057] a) AC16 cells treated in step (1) were plated into a 96-well plate, with 1×10 cells per well. 4 The cells were divided into different MOI (without polybrene) experimental groups, different MOI (with polybrene) experimental groups and Blank cell group. The MOI was set to 0, 10, 20, and 40. The culture medium used was DMEM (high glucose) culture medium.

[0058] b) The next day, the cell status was observed. When the cells were in good growth condition and the confluence reached 60-70%, the culture medium was discarded, and a corresponding volume of lentivirus was added, and DMEM (high glucose) medium or DMEM (high glucose) medium containing Polybrene was used for infection according to the experimental group.

[0059] The concentration of Polybrene in the culture medium is 5 μg / mL. The amount of virus added to each well = MOI × number of cells at infection / titer (TU / mL) × 10 3 The lentivirus was pSLenti-CMV-jGCaMP7f-PGK-Puro-WPRE (Heyuan Biotechnology).

[0060] c) 16 hours after infection, the cells were observed under a microscope, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture.

[0061] d) After 48 hours of infection, the cell status was observed under a microscope, and three fields of view were randomly selected for photography to verify the infection efficiency. The optimal infection condition was determined to be MOI = 20 (with Polybrene). The results are shown in Figure 1 shown.

[0062] Among them, the inverted fluorescence microscope selects FITC filter, the excitation wavelength is 490nm, and the emission wavelength is 520nm.

[0063] (3) Lentivirus infection of human cardiomyocyte cell line AC16

[0064] a) Human cardiomyocyte cell line AC16 cells were plated into 96-well plates, with 1×10 cells per well. 4The culture medium used was DMEM (high glucose).

[0065] b) The next day, observe the cell status. When the cell growth status is good and the confluence reaches 60-70%, discard the culture medium, calculate the amount of virus added to each well according to the optimal MOI = 20 (with Polybrene) determined in step (2), and add DMEM (high glucose) culture medium containing the corresponding volume of lentivirus and Polybrene for infection. The concentration of Polybrene in the culture medium is 5 μg / mL; the amount of virus added to each well = MOI × number of cells / titer at infection (TU / mL) × 10 3 .

[0066] c) 16 hours after infection, the cells were observed under a microscope, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture.

[0067] (4) Calcium ion fluorescence signal detection

[0068] After 48 hours of infection, the cells were placed under an inverted fluorescence microscope for observation. Among them, the inverted fluorescence microscope selected a FITC filter, with an excitation wavelength of 490 nm and an emission wavelength of 520 nm. Three fields of view were randomly selected for shooting. ImageJ software was used to quantitatively analyze the fluorescence intensity of the image, and the statistical data was used to quantitatively characterize the calcium level of myocardial cells based on the statistical results (ZhengQ, et al. Calcium transients on the ER surface trigger liquid-liquid phaseseparation of FIP200 to specify autophagosome initiation sites. Cell. 2022Oct27; 185(22): 4082-4098.e22.).

[0069] (5) Accuracy verification

[0070] In order to determine the accuracy of this method, a small molecule compound HBST271 (structural formula: Figure 2 This compound is known to have no agonistic effect on PIEZO1.

[0071] AC16 cells were divided into 3 groups. Control group (Con): simple lentivirus infection. Agonist group (Yoda1): lentivirus infection, followed by stimulation with PIEZO1 agonist Yoda1 (20 μM) for 24 h. Small molecule compound group (HBST271): lentivirus infection, followed by stimulation with small molecule compound (20 μM) for 24 h.

[0072] The cells were placed under an inverted fluorescence microscope for observation. Three fields of view were randomly selected for photography. ImageJ software was used to quantitatively analyze the fluorescence intensity of the images and to calculate the statistics. The results are shown in Tables 1 and Figure 3 shown.

[0073] Table 1 Relative fluorescence intensity of negative control

[0074]

[0075] The results showed that the relative fluorescence intensity of the PIEZO1 channel agonist Yoda1 was significantly stronger than that of the blank control group, while the relative fluorescence intensity of the negative control group had no significant difference from that of the blank control group. This shows that the activation of the PIEZO1 channel can enhance the level of calcium signals in cardiomyocytes, indicating that this method can not only detect calcium signals in cardiomyocytes, but also determine the stimulatory or inhibitory effects of compounds on PIEZO1.

[0076] Example 2 Detection of the agonistic effect of small molecule compounds on AC16 PIEZO1 channel

[0077] The small molecule compound is a substituted benzyl thioether oxadiazole compound, which may have a regulatory effect on the PIEZO1 ion channel, can specifically excite or inhibit the function of the PIEZO1 ion channel, has low potential cardiac toxicity, and has less toxic and side effects.

[0078] The structures of the above compounds are shown in Table 2.

[0079] Table 2 Preferred compound numbers and corresponding structural formulas

[0080]

[0081]

[0082] In order to determine whether the above small molecule compound is an agonist of the PIEZO1 channel, the method described in Example 1 was used to divide AC16 cells into the following three groups for experiment.

[0083] (i) Control group: simple lentivirus infection, same as Example 1.

[0084] (ii) Agonist group: lentiviral infection. After 16 h of infection, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture until 48 h of infection. The PIEZO1 agonist Yoda1 (20 μM) was added, and fluorescence microscopy was performed after 24 h of stimulation.

[0085] (iii) Small molecule compound group: The difference is that after 16 hours of infection, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture. After 48 hours of infection, the small molecule compound to be tested (20 μM) was added, and fluorescence microscopy observation was performed after 24 hours of stimulation.

[0086] The cells were observed under an inverted fluorescence microscope. Three fields of view were randomly selected for photography. ImageJ software was used to quantitatively analyze the fluorescence intensity of the images and to generate statistical data.

[0087] Small molecules whose relative fluorescence intensity increased by 20% or more compared with the control group were listed as possible agonists of the PIEZO1 channel.

[0088] The above compounds I-1 to I-18 were divided into three batches to test the agonist effect of the compounds at a concentration of 20 μM on PIEZO1. The results of the three groups are as follows.

[0089] (1) Group I compounds

[0090] The compounds were tested for agonism, and the results are shown in Table 3 and Figure 4 shown.

[0091] Table 3 Relative fluorescence intensity of the first batch of compounds

[0092]

[0093] In the agonist effect test of the first group of compounds, the fluorescence intensity of I-3 increased by 61.68%, which was better than Yoda1 (33.95%). The fluorescence intensity of I-2 increased by 31.18%, which was comparable to Yoda1.

[0094] (2) The second group of compounds

[0095] The compounds were tested for agonism, and the results are shown in Table 4 and Figure 5 shown.

[0096] Table 4 Relative fluorescence intensity of the second group of compounds

[0097]

[0098] In the agonist effect test of the second group of compounds, no compound had a superior agonist effect.

[0099] (3) The third group of compounds

[0100] The compounds were tested for agonism, and the results are shown in Table 5 and Figure 6 shown.

[0101] Table 5 Relative fluorescence intensity of the third group of compounds

[0102]

[0103]

[0104] In the agonist effect test of the third group of compounds, the fluorescence intensity was significantly increased in: I-5 (75.72%), I-6 (39.91%), I-8 (70.54%), I-16 (26.87%) and I-17 (40.26%), but they were all weaker than Yoda1 (139.79%).

[0105] In summary, with the fluorescence intensity increased by 20% compared with the control group as the standard, compounds I-2, I-3, I-5, I-6, I-8, I-16 and I-17 have better agonist effects, and the agonist effect of I-3 is better than that of Yoda1.

[0106] Example 3 Detection of the inhibitory effect of small molecule compounds on AC16 Peizo1 channel

[0107] According to the results of Example 2, compounds with no significant difference compared with the control group were selected to continue the inhibition test.

[0108] To determine whether the small molecule compounds are inhibitors of the PIEZO1 channel, AC16 cells were divided into two groups.

[0109] (i) Control group: lentiviral infection. After 16 h of infection, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture until 48 h of infection. The PIEZO1 agonist Yoda1 (20 μM) was added, and fluorescence microscopy was performed after 24 h of stimulation.

[0110] (ii) Small molecule compound group: lentivirus infection. After 16 h of infection, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture until 48 h of infection. PIEZO1 agonist Yoda1 (20 μM) and small molecule compounds (20 μM) were added at the same time. Fluorescence microscopy was performed after 24 h of stimulation.

[0111] The cells were observed under an inverted fluorescence microscope. Three fields of view were randomly selected for photography. ImageJ software was used to quantitatively analyze the fluorescence intensity of the images and statistically analyze the data. Small molecules whose relative fluorescence intensity decreased by 20% or more compared with the control group were listed as possible inhibitors of the PIEZO1 channel.

[0112] (1) Group I compounds

[0113] The compounds in Table 3 that had no significant difference compared with the control group were selected for inhibitory effect detection, and the results are shown in Tables 6 and Figure 7 shown.

[0114] Table 6 Relative fluorescence intensity of the first group of compounds

[0115]

[0116] I-1, whose fluorescence intensity of agonistic effect was not significantly different from that of negative control, was selected to test the fluorescence intensity of inhibitory effect, and the fluorescence intensity was reduced by 28.87%.

[0117] (2) The second group of compounds

[0118] The compounds in Table 4 that had no significant difference compared with the control group were selected for inhibitory effect detection, and the results are shown in Tables 7 and Figure 8 shown.

[0119] Table 7 Relative fluorescence intensity of the second group of compounds

[0120]

[0121] Small molecule compounds with no significant difference in fluorescence intensity between the agonist effect and the negative control were selected to test the inhibitory effect. The inhibitory effects were as follows: I-10 fluorescence intensity decreased by 22.13%, I-14 fluorescence intensity decreased by 24.95%, and I-13 fluorescence intensity decreased by 23.04%.

[0122] (3) Effect diagram of the third group of compounds

[0123] The compounds in Table 5 that had no significant difference compared with the control group were selected for inhibitory effect detection, and the results are shown in Tables 8 and Fig. 9 shown.

[0124] Table 8 Relative fluorescence intensity of the third group of compounds

[0125]

[0126] Compounds whose fluorescence intensity of agonist effect was not significantly different from that of negative control were selected to test the inhibitory effect. If the relative fluorescence intensity did not change much, it meant that there was no inhibitory effect.

[0127] In summary, compounds I-1, I-10, I-13 and I-14 had an inhibitory effect, taking a 20% decrease in fluorescence intensity compared with the control group as the standard.

[0128] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitution combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of the present invention.

Claims

1. Application of a calcium ion indicator GCaMP in the detection of calcium signals in human cardiomyocytes.

2. Application of a lentivirus packaged with the calcium ion indicator GCaMP in the detection of calcium signals in human cardiomyocyte cell lines.

3. A method for detecting calcium signals in human cardiomyocytes using a lentivirus packaged with the calcium ion indicator GCaMP.

4. The method according to claim 3, characterized in that It includes the following steps: (1) Recovery, culture, digestion and subculture of human cardiomyocytes; (2) lentiviral infection of human cardiomyocyte cell lines; (3) 48 h after infection, the cells were observed under a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed and statistically analyzed.

5. The method according to claim 4, characterized in that Step (2) specifically includes: a) Plating the human cardiomyocytes treated in step (1) into a well plate; b) Calculate the amount of lentivirus added to each well according to the most suitable multiplicity of infection, and prepare the infection solution with DMEM high-glucose medium containing 5 μg / mL Polybrene; c) Discard the old culture medium in the well plate and add the infection solution; after 16 hours of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-glucose culture medium, and continue to culture until infection for 48 hours.

6. The method according to claim 5, characterized in that The optimal MOI in step (2) is 20.

7. The method according to claim 4, characterized in that The lentivirus is pSLenti-CMV-jGCaMP7f-PGK-Puro-WPRE.

8. Use of a lentivirus packaged with the calcium ion indicator GCaMP in detecting the agonist and / or inhibitory effect of a compound on the PIEZO1 channel of a human cardiomyocyte cell line.

9. A method for detecting the agonist and / or inhibitory effect of a compound on the PIEZO1 channel of a human myocardial cell line, characterized in that: It includes the following steps: (1) Recovery, culture, digestion and subculture of human cardiomyocyte cell line AC16; (2) lentivirus infection of human cardiomyocyte cell line AC16 treated in step (1); I) Plating the human cardiomyocytes AC16 treated in step (1) into a well plate; II) Divide the plate into two groups and perform lentiviral infection separately: (i) Control group A: Calculate the amount of lentivirus per well according to MOI = 20, and prepare the infection solution with DMEM high-glucose medium containing 5 μg / mL Polybrene; discard the old medium in the well plate and add the infection solution; after 16 hours of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-glucose medium, and culture until infection for 48 hours; continue to culture for 24 hours; (ii) Experimental group A: The amount of lentivirus added to each well was calculated according to MOI = 20, and the infection solution was prepared with DMEM high-glucose medium containing 5 μg / mL Polybrene; the old medium in the well plate was discarded and the infection solution was added; after 16 hours of infection, the infection solution was discarded, PBS was washed twice, and fresh DMEM high-glucose medium was added, and cultured until infection for 48 hours; 20 μM of the small molecule compound to be tested was added and cultured for another 24 hours; (3) Observe under a fluorescence microscope and perform quantitative analysis and statistical data on the fluorescence intensity; The relative fluorescence intensity of the experimental group A relative to the control group A is obtained, and the small molecule compound to be tested that increases by more than 20% compared with the control group is an agonist of the PIEZO1 channel; (4) selecting small molecule compounds to be tested whose relative fluorescence intensity of experimental group A is not significantly different from that of control group A; (5) lentivirus infection of human cardiomyocyte cell line AC16 treated in step (1); I) Plating the human cardiomyocytes AC16 treated in step (1) into a well plate; II) Divide the plate into two groups and perform lentiviral infection separately: (i) Control group B: Calculate the amount of lentivirus per well according to MOI=20, and prepare the infection solution with DMEM high-glucose medium containing 5 μg / mL Polybrene; discard the old medium in the well plate and add the infection solution; after 16 hours of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-glucose medium, and culture until infection for 48 hours; add 20 μM Yoda1 and continue to culture for 24 hours; (ii) Experimental group B: Calculate the amount of lentivirus to be added to each well according to MOI = 20, and prepare the infection solution with DMEM high-glucose medium containing 5 μg / mL Polybrene; discard the old medium in the well plate and add the infection solution; after 16 hours of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-glucose medium, and culture until infection for 48 hours; add 20 μM Yoda1 and 20 μM of the small molecule compound to be tested obtained by step (4) and continue to culture for 24 hours; (6) Observe under a fluorescence microscope, perform quantitative analysis on the fluorescence intensity, and collect statistical data; The relative fluorescence intensity of the experimental group B relative to the control group B is obtained, and the small molecule compound to be tested that decreases by more than 20% compared with the control group is an inhibitor of the PIEZO1 channel.

10. The method according to claim 9, characterized in that In the step (4), a small molecule compound to be tested whose relative fluorescence intensity of the experimental group A is 80% to 120% of that of the control group A is selected.

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