Biophysical properties of NaV1.5 channels from atrial-like and ventricular-like cardiomyocytes derived from human induced pluripotent stem cells – Scientific Reports

hiPSC cultures and cardiomyocyte differentiation

The hiPSC lines CBRCULi001-A⁵⁴ and CBRCULi008-A⁵⁵ were generated from a 44-year-old male and 75-year-old female control lymphoblastoids, respectively, and they were reprogramed at the LOEX core facility (Quebec City, QC, Canada). All the work with hiPSCs were approved by CIUSSS de la Capitale-Nationale ethics committee (Project #2019-1734). All methods were carried out in accordance with relevant guidelines and regulations and were conducted by respecting approved protocols by the ethics committee. Furthermore, these procedures were only performed after obtaining informed consent from both patients. The hiPSC line CBRCULi008-A was used for all experiments and the hiPSC line CBRCULi001-A for experiments indicated in the supplementary data. They were grown on hESC-qualified Matrigel (Cat# 354277, Corning, AZ, USA) in mTeSR plus medium (Cat# 100-0276, STEMCELL Technologies, BC, Canada) and were routinely dissociated using 500 μM EDTA every 4–6 days. They were then differentiated into ventricular (vCMs) and atrial cardiomyocytes (aCMs). The hiPSCs were differentiated into ventricular cardiomyocytes (vCMs) with a monolayer-based protocol using STEMdiff™ Ventricular Cardiomyocyte Differentiation kits (Cat# 05010, STEMCELL Technologies) according to the manufacturer’s protocols and instructions. Spontaneously beating cells were observed at day 8 to 12 of differentiation. The hiPSCs were also differentiated into atrial cardiomyocytes (aCMs) by adding 1 μmol/L all-trans retinoic acid (RA) between 2 and 5 days of differentiation (Cat# 72262, STEMCELL Technologies). The hiPSC-CMs were maintained in STEMdiff™ Cardiomyocyte Maintenance medium (Cat# 05020, STEMCELL Technologies) until they reached 30 days of maturation. Only the efficient differentiations with a spontaneously beating monolayers, were selected to perform the experiments.

Gene expression analysis

RNA was extracted from the hiPSC-CMs on day 30 of differentiation using Quick-RNA MiniPrep kits (Cat# R1054, Cedarlane, ON, Canada), and cDNA was synthesized by the QuantiTect Rev. Transcription Kit protocol (Cat# 205313, Qiagen, Hilden, Germany). qPCR assays were performed using SYBR green I detection dye on an LC480 platform (Roche, Basel, Switzerland) using the seller’s specifications. The primers are listed in Supplementary Table S3. All the qPCR reactions were run in triplicate with a non-template control (NTC). qPCR efficiencies were obtained using a series of cDNA dilutions and were calculated using the slope of the regression line determined using the following equation: E = 10 [− 1/slope]. All qPCR reactions had an efficiency ranging from 1.7 to 2.3. The analyses were performed using LightCycler^® 480 SW 1.5 software. Run-to-run variations were adjusted using a known standard, and quantifications were corrected for efficiency. The specificity of the amplification for each run was controlled using a melting curve analysis. The quality of the differentiation was assessed by measuring the TNNT2 gene level. All the samples with the lowest cycle threshold (CT) values were totally removed. When comparing vCM and aCM conditions for a specific gene, normalization was carried out by dividing the CT of aCM by that of vCM. To facilitate comparisons across different genes, normalization was performed with respect to a single gene. Gene expression’s relative quantification was executed using the ΔΔCT method. For standardization purposes, the obtained relative abundance value was divided by the mean value obtained from two housekeeping genes (RPL22, PPIA). For the SCN5A mRNA analysis, targeting the exon 25 of SCN5A mRNA covered all isoforms, including the adult (exon 6b) and “neonatal” (or fetal, exon 6a) sodium channel isoforms. The percentage of adult isoform was obtained using the following ratio: isoform adult (exon 6b)/isoform neonatal (exon 6a) × 100.

Western blotting

The proteins of the hiPSC-CMs were extracted on day 30 of differentiation by scraping the cells into Radioimmunoprecipitation Assay buffer (RIPA buffer: 50 mmol/L Tris–Cl, 1 mmol/L EDTA, 150 mmol/L NaCl, 0.5% SDS, 1% NP-40) supplemented with proteases (Cat# 5892970001, Sigma-Aldrich) and phosphatase inhibitor cocktails (Cat#4906845001, Sigma-Aldrich). The lysate was incubated for 2 h at 4 °C under gentle rotation and was clarified by centrifugation at 18,000 g for 5 min at 4 °C. Protein concentrations were measured using Pierce™ BCA Protein Assay kits (Cat# 23225, ThermoFisher Scientific) with a bovine serum albumin (BSA) standard range (20 to 2000 µg/mL) as a reference. Protein extracts (20 μg) were denatured in 5X sample buffer (156 mM Tris–Cl, pH 6.8, 0.025% bromophenol blue, 5% SDS, 50% glycerol, 12.5% β-mercaptoethanol) at 37 °C for 30 min. They were resolved on 4–15% Mini-PROTEAN^® TGX Stain-Free™ Protein gels (Cat# 456-8083, Bio-Rad) and were blotted on 0.45-μm PVDF membranes with Trans-Blot Turbo RTA Midi 0.45 µm LF PVDF Transfer kits (Cat# 1704275, BioRad). The PVDF membranes were blocked and were incubated with rabbit anti-sodium voltage-gated channel alpha subunit 5 (SCN5A) (1:200, Cat# ASC-005, Alomone Labs, RRID:AB_2040001), rabbit anti-calcium voltage-gated channel subunit alpha 1 C (CACNA1C) (1:200, Cat# ACC-003, Alomone Labs, RRID:AB_2039771), mouse anti-myosin light chain 7 (MYL7) (1:400, Cat# ab68086, Abcam, RRID:AB_1140497), rabbit anti-myosin light chain 2 (MYL2) (1:2000, Cat# ab79935, Abcam, RRID:AB_1952220), mouse anti-TNNT2 (1:5000, Cat# ab10214, Abcam, RRID:AB_2206574), rabbit anti-gap junction protein alpha 1 (GJA1) (1:5000, Cat# ab11370, Abcam, RRID: AB_297976), rabbit anti-potassium voltage-gated channel subfamily A member 5 (KCNA5) (1:200, Cat# APC-150, Alomone Labs, RRID: AB_10918640), rabbit anti-ryanodine receptor 2 (RYR2) (1:1000, Cat# ARR-002, Alomone Labs, RRID: AB_2040184), or rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:20,000, Cat# A300-641A, Bethyl, RRID:AB_513619). Goat horseradish peroxidase (HRP)-conjugated anti-rabbit (1:10,000, Cat# 111-035-003, Jackson ImmunoResearch, RRID:AB_2313567) and anti-mouse (1:10,000, Cat# 115-035-003, Jackson ImmunoResearch, RRID:AB_10015289) were used as secondary antibodies. Proteins were revealed using Clarity and Clarity Max Western ECL substrates(Cat# 1705060 and Cat# 1705062, Bio-Rad) and were visualized using the ChemiDoc system (Bio-Rad, ON, Canada). Relative quantification of protein expression was performed with ImageJ software. The total intensities of the bands of the targeted proteins were divided by the normalization factor obtained after dividing the observed signals of total protein in the lane with the strongest observed signal of total protein on the blot. Images of all the gels and blots are available in Suppl. Fig. S4.

Immunocytofluorescence staining

hiPSC-CMs were dissociated at D23 using STEMdiff™ Cardiomyocyte Dissociation Medium (Cat# 05025, STEMCELL Technologies) and were seeded on Matrigel-coated 12-mm glass coverslips in a 24-well plate at a density of 50,000 cells/cm². At D30, the cells were fixed with 4% paraformaldehyde, permeabilized, and saturated in a PBS solution containing 0.2% Triton X-100, 5% goat serum, and 1% BSA for 30 min. hiPSC-CMs were stained overnight at 4 °C using a blocking solution (1% BSA / 5% GS in PBS) containing the following primary antibodies: human anti-TNNT2-FITC (1:200, Cat# 130-119-674, Miltenyi Biotec, RRID:AB_2751795), mouse anti-actinin alpha 1 (ACTN1) (1:500, Cat# ab9465, Abcam, RRID:AB_307264), rabbit anti-GJA1 (1:500, Cat# ab11370, Abcam, RRID:AB_297976), rabbit anti-MYL2 (1:100, Cat# ab79935, Abcam, RRID:AB_1952220), and human anti-MYL7-PE (1:20, Cat# 130-117-546, Miltenyi Biotec, RRID:AB_2751399). The secondary antibodies (goat anti-mouse Cy3 (1:500, Cat# A10521, Invitrogen, RRID:AB_2534030) and goat anti-rabbit AlexaFluor™ 633 (1:500, Cat# A21071, Invitrogen, RRID:AB_2535732) were then added to the blocking solution. The mixtures were incubated for 2 h at room temperature in the dark. After washing, the glass coverslips were slide mounted in Fluoromount-G mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Cat# 00-4959-52, Invitrogen). The immunolabeled samples were acquired at × 20 objective using a Zeiss LSM780 confocal laser scanning microscope (Zeiss, Germany) and examined with ZEN software (Zeiss, Germany) and adapted with ImageJ software (NIH, Bethesda, MD, USA).

Electrophysiology

Patch-clamp experiments were performed at room temperature using an Axopatch 200B amplifier and pClamp software v10 (Molecular Devices, CA, USA). Macroscopic Na⁺, Ca²⁺ and K⁺ currents and APs were recorded using the whole-cell configuration of the patch-clamp technique in voltage-clamp and current-clamp modes, respectively. The pipettes were drawn from borosilicate glass capillaries (Sutter Instrument, CA, USA) and were fire polished.

For the voltage-clamp experiments, the pipettes were coated with HIPEC (Dow-Corning, MI, USA) to minimize electrode capacitance. For Na⁺ currents, the pipettes were filled with a solution containing (in mmol/L) 35 NaCl, 105 CsF, 10 EGTA, and 10 HEPES. The pH was adjusted to 7.4 with CsOH. The bath solution contained (in mmol/L) 105 NMDG, 35 NaCl, 2 KCl, 1.5 CaCl₂, 1 MgCl₂, 10 D-glucose, 10 HEPES, 10 TEA-Cl, and 0.01 nifedipine⁵⁶. The pH was adjusted to 7.4 with methanethiosulfonic (MTS) acid. For Ca²⁺ currents, the pipettes were filled with a solution containing (in mmol/L) 25 NaCl, 105 CsCl, 1 MgCl₂, 10 EGTA, and 10 HEPES. The pH was adjusted to 7.2 with CsOH. The bath solution contained (in mmol/L) 100 NaCl, 5 CsCl, 5 CaCl₂, 40 NMDG, 1 MgCl₂, 10 D-glucose, 10 HEPES, and 15 TEA-Cl. The pH was adjusted to 7.4 with methanesulfonic acid (MSA). For K⁺ currents, the pipettes were filled with a solution containing (in mmol/L) 5 NaCl, 5.4 KCl, 136 KMeSO₄, 5 EGTA, 5 MgATP, 5 Phosphocreatine, 1 MgCl₂, and 1 HEPES. The pH was adjusted to 7.2 with KOH. The bath solution contained (in mmol/L) 136 NaCl, 5.4 KCl, 1.8 CaCl₂, 1 MgCl₂, 0.33 NaH₂PO₄, 5 HEPES, and 10 D-glucose. The pH was adjusted to 7.4 with NaOH. 20 µmol/L Tetrodotoxin (Cat# L8503, Latoxan, France), and 10 µmol/L Nifedipine (Cat# N7634, Millipore Sigma) were added to inhibit voltage-gated sodium channels and voltage-gated calcium channels, respectively. To study the effect of 4-aminopyridine (4-AP, Cat# 104570050, ThermoFisher Scientific) on K⁺ currents, 4-AP was added in the bath solution and perfused directly on the hiPSC-CM with a 4-channel perfusion system (Automate Scientific). A low concentration of 4-AP was used on aCMs (0.1 mmol/L) and a higher dose on vCMs (1 mmol/L). 4-AP-sensitive currents were obtained after the subtraction of K⁺ currents recorded under 4-AP treatment to K⁺ currents recorded without 4-AP treatment. Series resistance and cell capacitance were corrected. Currents were filtered at 5 kHz, digitized at 10 kHz, and stored on a microcomputer equipped with an AD converter (Digidata 1440A, Molecular Devices). P/4 leak subtraction was used prior to applying pulse stimulations for Na⁺ and Ca²⁺ current recordings.

For I_f currents, the pipettes were filled with a solution containing (in mmol/L) 130 K-aspartate, 10 NaCl, 10 HEPES, 5 EGTA, 2 CaCl₂, 2 MgCl₂, 0.1 Na-GTP, 5 Na₂-GTP, 5 Phosphocreatine. The pH was adjusted to 7.2 with KOH. The bath solution contained (in mmol/L) 140 NaCl, 5.4 KCl, 5 HEPES, 1.8 CaCl₂, 1 MgCl₂, 1 BaCl₂, 2 MnCl₂, 5.5 D-Glucose. The pH was adjusted to 7.4 with NaOH.

Na⁺ currents were obtained using 200-ms pulses from − 100 to + 60 mV in + 5 mV increments. Ca²⁺ currents were obtained using 250-ms pulses from − 40 to + 60 mV in + 5 mV increments. K⁺ currents were obtained using 300-ms pulses from − 80 to + 20 mV in + 10 mV increments. I_f currents were obtained using 10-s pulses from − 35 to -125 mV in − 10 mV increments, followed by a 1-s pulse at − 125 mV. The densities were measured by normalizing current amplitudes to membrane capacitance. Activation curve for I_f currents was determined from the tail currents. Activation curves for Na⁺, Ca²⁺ and K⁺ currents were generated using the following standard Boltzmann distribution:

Inactivation Na⁺ currents were obtained using 20-ms test pulses to − 30 mV after a 500-ms pre-pulse to potentials ranging from − 120 to + 30 mV. Inactivation Ca²⁺ currents were obtained using 30-ms test pulses to + 10 mV after a 3000-ms pre-pulse to potentials ranging from − 40 to + 20 mV. The inactivation values were fitted to the following standard Boltzmann equation:

For Na⁺ recovery from inactivation, cells were depolarized to − 30 mV for 40 ms from a holding potential of − 100 mV to inactivate the Na⁺ channels. Test pulses were then applied at − 30 mV for 20 ms to measure current amplitudes, with intervals ranging from 0.06 to 4000 ms. The resulting curves were fitted with a double exponential equation. The time constants of fast Na⁺ and Ca²⁺ inactivation decay, were plotted as a function of voltage. The time constants were obtained using a simple exponential function. Window current was obtained thank to activation and inactivation parameters with the following formula:

For the current-clamp experiments, the patch pipettes (resistance 2–5 mΩ) were filled with a solution containing (in mmol/L) 10 NaCl, 122 KCl, 1 MgCl₂, 1 EGTA, and 10 HEPES. The pH was adjusted to 7.3 with KOH. The bath solution was composed of (in mmol/L) 154 NaCl, 5.6 KCl, 2 CaCl₂, 1 MgCl₂, 8 D-glucose, and 10 HEPES. The pH was adjusted to 7.3 with NaOH. The APs were recorded at a 1-Hz stimulation frequency. The holding potential during the recordings was maintained at − 80 mV with the holding command mode of the amplifier or with a Cybercyte V10 dynamic clamp system (Suppl. Fig. S5) (Cytocybernetics, North TonaWanda, NY, USA). The duration of the stimulation pulse was 3 ms with 0.2- to 1.5-nA injected currents depending on the cell. The recorded resting membrane potential (RMP) was measured before establishing the holding potential of − 80 mV and before initiating the 1-Hz stimulation. To perform a classification of cell subtypes as ventricular-, atrial-, and nodal-like cardiomyocytes, we used an approach based on AP duration⁵⁷. Cells exhibiting an action potential duration at 90% repolarization (APD₉₀) exceeding 250 ms were automatically categorized as ventricular-like, while those with an APD₉₀ shorter than 250 ms were classified as either atrial-like or nodal-like. To distinguish between atrial-like and nodal-like cells, we employed a criterion based on the difference between the APD₅₀ and APD₂₀ when stimulated at a frequency of 1 Hz. Cells displaying a difference less than 10 ms (APD₅₀–APD₂₀ ≤ 10 ms) were designated as nodal-like, whereas those surpassing this threshold were labeled as atrial-like. This particular criterion serves to emphasize the distinct AP profiles exhibited by nodal-like cells. We used a total of four independent differentiations to collect the electrophysiological data for the first cell line of iPSC-CMs, and three independent differentiations for the second cell line. These multiple independent differentiations allowed us to obtain robust and reliable electrophysiological measurements for our analysis.

Optical mapping

vCMs and aCMs were dissociated at day 12 to 15, and cardiac monolayers were generated by seeding hiPSC-CMs (350,000 cells) on hESC-qualified Matrigel-coated (Cat# 07181, Corning) 13-mm TC coverslips (Cat# 83.1840.002, Sarstedt) in a 24-well plate. hiPSC-CM monolayers were cultured in STEMdiff™ Cardiomyocyte Maintenance Medium until used (30–40 days of maturation). For dual mapping, the monolayers were first stained with the Ca²⁺ indicator Rhod-2 AM (5 µmol/L, Cat# ab142780, Abcam) and incubated at 37 °C for 30 min, washed, loaded with the potentiometric dye RH 237 (15 µmol/L, Cat# S1109, ThermoFisher), and incubated for an additional 30 min. The cultures were then placed in an imaging solution containing (in mmol/L) 154 NaCl, 5.6 KCl, 2 CaCl₂, 1 MgCl₂, 8 D-glucose, and 10 HEPES, pH 7.3). The optical mapping system used to record AP and Ca²⁺ transients at 500 frames per second is an epifluorescence macroscope equipped with two CMOS N256 cameras (MiCAM03, Brainvision, SciMedia Ltd., USA), a 530 nm green LED light source (LEX2-LZ4-G), an imaging cube containing a collimator, a dichroic mirror (560 nm), and a bandpass excitation filter (50 mm, BrightLine^®, Semrock). A second imaging cube was used as a beam splitter and contained a 662-nm dichroic mirror, two emission filters (572/15 nm, BrightLine^®, Semrock), a longpass filter (715 nm, Andover Corporation), and a lens system with a maximum aperture of f/1.4 for RH 237 and Rhod-2 imaging.

The hiPSC-CMs were kept at 37 °C using a controlled heating plate (Multi Channel Systems, Reutlingen, Germany) and were electrically paced by two platinum/iridium electrodes positioned at the lower edge of the monolayers. A stimulus generator (STG4002, Multichannel Systems) was used to deliver 10-ms square bipolar pulses at a frequency of 1 Hz. Before adding blebbistatin (10 µmol/L, Cat# 72402, STEMCELL Technologies) to the imaging solution to prevent contraction artifacts, spontaneous electrical activity was recorded. Thereafter, all monolayers successfully paced at 1 Hz were analyzed. Raw optical signals were then processed, and were analyzed with Brainvision Workbench software (Brainvision, SciMedia) to extract the electrophysiological measurements, calculate the conduction velocities, and generate the activation maps.

Statistical analysis

All statistical analyses were performed using PRISM10 software (GraphPad, CA, USA). The normality of the distribution was determined using the D’Agostino-Pearson normality test. The data are expressed as median ± quartiles (25% and 75%) with min to max values or as mean ± SEM (standard error of the mean). For the electrophysiological data, a replicate (n) represented the number of cells recorded. For the qPCR and Western blot data, a replicate (n) represented an RNA or protein extract from a single independent differentiation. For the optical mapping data, a replicate (n) corresponded to a hiPSC-CM monolayer in a 24-well plate. For two independent variables, such as the normalized intensity/voltage relationships (I/V) and the time constants of fast inactivation decay, a two-way ANOVA with a Šídák multiple comparisons test was used. Otherwise, a two-tailed unpaired Student’s t-test was used to compare aCM and vCM conditions. When three groups were compared (Fig. 5A), statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. When the replicate (n) was too small (Suppl. Fig. S1A), a nonparametric Mann–Whitney test was performed. The Brubbs’ test, available on the GraphPad website, was performed to identify and exclude outliers that significantly deviated from the overall trend. All the statistical tests were performed using a 95% confidence interval, and the differences were considered significant beyond the 0.05% risk threshold (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

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• Source: https://www.nature.com/articles/s41598-023-47310-6