Cell culture and cardiac differentiation of hESC
The hESC line was purchased from Cellapy (Beijing, China) was routinely maintained in the presence of PSCeasy medium (Cellapy, China) on six-well plates (Corning, USA) coated with 5% Matrigel (Corning, USA). Medium was changed every day and passaged every 2–3 days with EDTA (Cellapy, China). The cells were grown in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. The hESCs were differentiated when they reached 70–80% confluence. Medium was changed to the basal differentiation medium. For day 0 to day 2, medium was changed to the basal medium C01 (Cellapy, China). For day 2 to day 4, medium was changed to the basal differentiational medium C02 (Cellapy, China). On day 4, medium was changed to the basal differentiational medium C03 (Cellapy, China) and changed medium every other day. Contracting cells were noted from day 9.
KCNQ1 single-stranded guide RNA (sgRNA) (ATGCTACACGTCGACCGCCA,CAGCCGCCCCCAGAGGCCCA,GCTCGAGGAAGTTGTAGACG) was designed using an online tool (https://www.synthego.com). We electroporated the epiCRISPR vector and sgRNA (100 µl electrotransformation solution (Cellapy, China) plus 2.5 µg KCNQ2 gRNA plasmid) into the cells using the 4D nuclear receptor system and the CA137 programme (Lonza, Germany). The transfected cells were seeded in 6-well plates and cultured overnight in PSCeasy medium 10 μM of Rho kinase inhibitor Y-27632. The medium was changed the next day. Drug (puromycin) selection was initiated after 72 h of transfection at a lower concentration of 0.1 µg/ml for the first hour and then at 0.3 µg/ml until the transfected lines were stable. The surviving cells were collected in 48-well plates and amplified for polymerase chain reaction (PCR) screening. The point mutation cells were prepared by epi-ABEmax/epi-AncBE4max/epi-ABEmax-NG/epi-AncBE4 max-NG plasmid. The plasmid was transfected using Lipofectamine 3000, and then, the transfected cells were selected by blasticidin. The specific methods can refer to the previous research.
100 mM 293B, 100 µM isoproterenol (ISO), 5 µM propranolol, 100 µM amiodarone 100 µM MgCl2 (Selleck, USA) were diluted in C05 (Cellapy, China). hESC-CMs were treated with 293B, ISO, Propranolol for 12 h. hESC-CMs were treated with Amiodarone, MgCl2 for 30 min.
RNA extraction and RT-PCR
Total RNA from cells was extracted by using TRIZOL Reagent (Invitrogen, USA). An amount of 2 µg total RNA was reversed to cDNA by using the GoScript Reverse Transcription System (Promega, USA). Quantitative RT-PCR involved use of SYBR Green II (Takara, Japan) in the iQ5 system (Bio Rad, Hercules, CA). A comparative CT method was used to analyze the relative changes in gene expression. The results were expressed as relative to the data of GAPDH transcripts (internal control). Primer sequences are listed in Additional file 1: Table S1.
Immunofluorescent staining (IF) and imaging analyses
The cells were plated on 20 mm coverslips coated with 5% Matrigel and were fixed with 4% PFA for 15 min. Then, after washing with PBS three times for 5 min, the cells were permeabilized with 0.2% Triton X-100 (Sigma, USA) for 15 min and blocked with 3% BSA (Sigma, USA) for 1 h at room temperature. After that cells were incubated with primary antibodies, overnight at 4 °C. Then, cells were washed by PBS and incubated for 1 h at room temperature in the dark with secondary antibodies (Invitrogen, USA). Cells were washed again as above, mounted with Fluoroshield Mounting Medium with DAPI (4, 6 diamino-2-phenylindole). Images were taken under a Confocal Microscope (Leica DMI 4000B, German). The antibody and their appropriate dilution are provided in Additional file 1: Table S2.
Western blot (WB) analysis
Protein from hESC-CMs was extracted by using a Protein Extraction Kit (Promega, USA). The protein concentration of the supernatant was measured by BCA method. The 30 µg protein was separated on 10% SDS-PAGE and transferred to PVDF membrane at 300 mA for 90 min, which was blocked with 5% albumin bovine (BSA) at room temperature for 1 h, then incubated at 4 °C overnight with the primary antibodies, then with IR dye-conjugated secondary antibodies (LI-COR, USA) for 1 h at room temperature. GAPDH was used as an internal control. Blots were exposed and analyzed with use of an Odyssey infrared imaging system (LI-COR Biosciences, USA). The antibody and their appropriate dilution are provided in Additional file 1: Table S2.
The hESC-CMs under different treatments were singularized with CardioEasy Human Cardiomyocyte Digestive Fluid (Cellapy, China). Observe that most of the clones are detached from the bottom of the plate under the microscope, gently pipette the cells and suck them out, centrifuge, and wash three times with PBS. The cells were stained with different antibodies, filtered through the 300 mesh filter, and immediately analyzed by FACS (Beckman, USA). The cell count is generally 1–2 million. The results were analyzed with Flow Jo X program.
Microelectrode array (MEA) analysis
hESC-CMs were digested in CardioEasy Human Cardiomyocyte Digestive Fluid (Cellapy, China), after which 2 × 104 cells were plated on a microelectrode array (MEA) pre-coated with 5% Matrigel (Cellapy, China). The next day, 300 μl medium was added to each well. After the hESC-CMs resumed spontaneous beating, the experimental data were recorded on a Maestro EDGE (Axion Biosystems, Inc., Atlanta, USA) according to the MEA manual. Cardiac Analysis Tool, AxIS Navigator, AxIS data export tool, and Origin were used to analyze the data.
Results are expressed as mean ± SD. Statistical analysis was performed with GraphPad Prism 8.00 for Windows. Two-sided unpaired Student’s t test was used to compare 2 groups with normal distribution. One-way ANOVA was used to compare 3 or more groups. All tests for normality and homogeneity of variance were passed before t test and one-way analysis of variance. P values of less than 0.05 were used to denote statistical significance. *P < 0.05; **P < 0.01, ***P < 0.001; NS, not significant.