Generation of human iPS cell lines
The human iPS cell lines used in this study were derived from the peripheral blood mononuclear cells of two Brugada patients and one healthy volunteer. The approval of the study was provided by the Bioethics Committee of the First Affiliated Hospital of Nanjing Medical University (2014-SR-090). The iPSC lines were generated using a CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, A16517), which employed four Yamanaka factors (OCT4, KLF4, SOX2 and cMYC), following the manufacturer’s instructions with subtle adjustments. Reprogrammed PBMCs were replated onto Matrigel (Corning, Corning, NY, USA, 354277) coating plates, and the medium was changed to mTeSR™1 (STEMCELL Technologies Inc., Vancouver, Canada, 05850) after transduction. Individual colonies could be identified on day 7 and were picked 20 days later and were maintained in mTeSR™1 medium.
Genome sequencing
To confirm the presence of the variations in the two iPS cell lines, patient-derived iPSCs were examined by Sanger sequencing. Total DNA was isolated using a TIANamp Genomic DNA Kit (TIANGEN Biotech, BJ, China), and the coding regions of SCN5A and SCN1B, including each variation site, were amplified by polymerase chain reaction (PCR). The PCR products were purified using a DNA Purification Kit (TIANGEN Biotech) and then were sequenced. The results were compared with the SCN5A and SCN1B reference sequences NM_001099404 and NM_001037.
Differentiation of hiPSC-CMs
A monolayer-based cardiomyocyte differentiation protocol was applied as previously described. Briefly, iPSCs were allowed to grow to over 90% confluence in mTeSR™1 medium and then were switched to RPMI 1640 medium (Gibco, 1744361) containing B-27® supplement without insulin (Gibco, A1895601). On day 0–1, additional CHIR-99021 (6 µM, Selleckchem, S2924) was added to induce mesodermal differentiation. On days 3–4, IWR-1 (5 µM, Sigma-Aldrich, 10161) was added to the medium to inhibit Wnt signaling and induce cardiogenesis. From day 8 onward, cells were cultured in RPMI 1640 containing B27® complete supplement (Gibco, 17504-044), and the medium was refreshed every other day. Beating cardiomyocytes could be observed from approximately days 8 to 10. Cells were cultured for at least 8 weeks and were dissociated using 0.25% trypsin-EDTA for further functional and electrophysiological analyses.
Flow cytometry analysis
Cells were treated with the appropriate dissociation solution (Accutase for iPS cells, 2.5% trypsin for CMs) and fixed with 4% paraformaldehyde at room temperature for 20 min. Pluripotent stem cells were counted and stained using antibodies against SSEA-4 (1:50 dilution, phycoerythrin conjugated, Miltenyi Biotec, FAB1435P-025) for 30 min at 4℃ shielded from light. Cardiomyocytes were co-incubated with anti-cTnT-FITC (1:50, Miltenyi Biotec, 130-106-687) and anti-MLC2v-PE (1:50, Miltenyi Biotec, 130-106-133) in PBS at 4 °C for 30 min. REA Control-FITC (1:50, Miltenyi Biotec, 130-104-611) and REA Control-PE (1:50, Miltenyi Biotec, 130-104-613) were used as isotype controls. The cells were measured using FACSCalibur (BD, Franklin Lakes, NJ, USA) and subsequent analysis were performed using FlowJo software(Tree Star, version 10.5.3).
Immunofluorescence staining
Cells were seeded on a coverslips and cultured for 3–5 days before being fixed in 4% paraformaldehyde for 20 min. The cells were permeabilized with 0.1% Triton X-100 for 5 min, when needed. Cells were then blocked in 5% BSA in PBS for 30 min; all of these steps occurred at room temperature. Cells were then incubated overnight at 4 °C with primary antibodies. For pluripotent stem cells, anti-SSEA4 (1:200, Abcam, ab16287), anti-Oct4 (1:200, Abcam, ab200834), anti-Nanog (1:200, Abcam, ab109250) and TRA-1-81 monoclonal antibody (1:150, Thermo Fisher Scientific, MA1-024) were applied. For cardiomyocytes, anti-α-actinin (1:200, Sigma-Aldrich, A7811), anti-cardiac troponin T (1:200, Abcam, ab8295), anti-MLC2v (1:200, Abcam, ab79935) were used. The following secondary antibodies include Alexa Fluor® 488 goat anti-rabbit IgG (1:800, Abcam, ab150077) and Alexa Fluor® 555 goat anti-mouse IgG (1:800, Abcam, ab150118) were incubated with samples at room temperature for 30 min. The nuclei were stained with DAPI (Life Technologies, P36931). Then, the cells were rinsed with PBS and observed under a fluorescence microscope (Axio Imager A2, Carl Zeiss, Germany). Images were analyzed and merged with ImageJ (NIH, version 1.8.0_77).
Transmission electron microscopy (TEM)
Cardiomyocytes were prepared for TEM evaluation as described previously(15). Briefly, CMs were dissociated into single cells with 0.25% trypsin. Samples were then fixed with 2.5% glutaraldehyde (Sigma-Aldrich), post fixed with 1% osmium tetroxide and rinsed with PBS. After dehydration with a series of ethanol solutions and embedding into resin, ultrathin sections were cut with an ultramicrotome (Leica EM UC7; Leica, Wetzlar, Germany) and stained with lead citrate. Visualization was performed under a transmission electron microscope (JEM-1010, Jeol Ltd., Tokyo, Japan) equipped with a CCD camera operated at 75 kV.
Patch clamp analysis
Three to four days after dissociation, single cell patch clamp experiments were performed using an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA) and analyzed with Clampfit 10.3 software (Molecular Devices, Sunnyvale, CA). Glass pipettes were pulled using borosilicate glass (Sutter Instrument Co, Novato, CA) and a micropipette puller (Model P-100, Sutter Instruments, Novato, CA, USA). Pipettes with resistance ranging from 2–4 MΩ were perfused with specified intracellular solutions and were used.
Na measurement
The sodium currents (INa) of cardiomyocytes were recorded using a whole-cell patch-clamp technique at room temperature as previously described(8). Data were filtered at 2 kHz and acquired at 20 kHz. INa was examined using the following intracellular solution designed to eliminate the outward potassium current: 60 mM CsCl, 60 mM CsF, 10 mM TEA-Cl, 1 mM CaCl2, 1 mM MgCl2, 10 mM EGTA, 10 mM glucose, and 10 mM HEPES (pH 7.2 with CsOH). The external bath solution composition was as follows: 130 mM choline chloride, 10 mM NaCl, 1 mM TEA-Cl, 1.8 mM MgCl2, 1.8 mM CaCl2, 0.2 mM NiCl, 10 mM glucose, and 10 mM HEPES (pH 7.2 with NaOH). Nifedipine was added to exclude inward calcium current. The program for recording INa ranged from − 80 to + 40 mV in 10 mV increments that occurred every 50 ms. Cell capacitance was measured to normalize Ina by applying a 10 mV voltage step from − 100 mV, and current density was calculated as peak current divided by cell capacitance (pA/pF).
AP measurement
Spontaneous action potentials were recorded at 37 °C in Tyrode’s solution as previously described (16). Data were filtered at 5 kHz and digitized at 40 kHz. Pipettes were filled with the following internal solution (in mM): 150 KCl, 5 NaCl, 2 CaCl2, 5 MgATP, 5 HEPES, 5 EGTA, and 10 glucose (pH 7.2 with KOH). APs were characterized by resting membrane potential (RMP), maximum AP amplitude (APA), maximum upstroke velocity (Vmax) and average action potential duration at 90%, 50%, and 30% repolarization (APD90, APD50, APD30, respectively). Each average data was from 10 consecutive recorded APs.
Calcium transient
Cells were loaded with the calcium-sensitive dye fluo-4 AM (Life Technologies, F14201) at a working concentration of 5 µM for 60 min at 37 °C. After rinsing the iPSC-CMs, they were maintained in Tyrode’s solution. The spontaneous calcium fluorescence signal was recorded by an inverted fluorescence microscope (Axio Vert. A1, Carl Zeiss, Jena, Germany) equipped with an sCMOS camera (Tucsen, Dhyana95, China). The excitation and emission wavelengths of Fluo-4 were 488 nm and 505 nm, respectively. Recordings were obtained at the acquisition rate of 25 frames/second for a 15 second duration. To calibrate the cell-to-cell variability and background disturbance, the fluorescence intensity in the region of interest was determined based on the formula: (F-F0)/F0. Herein, F0 represents the baseline intensity, while F represents the intensity at any point in time. The parameters that describe and evaluate the calcium transient include the spontaneous beat rate, amplitude and arrhythmic events that are EAD-like or fibrillation-like. Data were analyzed using ImageJ as previously described(15).
Drug response
Quinidine (Selleckchem, S4658) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mM stock solution. Working solutions were freshly prepared in cell-specific medium. Final concentration was set as 0.1-fold to 10-fold the ETPC (unbound effective therapeutic plasma concentration) of quinidine (0.3, 1, 3,10, 30 µM). Dilutions ratio up to 1000 had no effect on cell apoptosis or cardiac electrical activity.
Baseline AP was recorded for each cell before the treatment. Then, quinidine was sequentially applied from the lowest to highest concentration at 10-min intervals for washout to obtain a steady state. For each concentration, 10 recorded APs from each cell were averaged for analysis. The following parameters were quantitated as mentioned above: RMP, APA, Vmax, APD30, APD50 and APD90. Recordings at each concentration were normalized to their baseline.
Statistical Analysis
Data with a normal distribution were presented as the mean ± SEM. One-way ANOVA was used to compare parameters among the test groups (i.e. the control, SCN1B, and SCN5A groups). Chi-square test was used for categorical variables. For pharmaceutical experiments, the post-drug effects were evaluated by comparing to the baseline using a paired Student’s t test. All statistical analyses were performed with R (version 4.0.2) and significance was determined at a P < 0.05.