NaV1.5 knockout in iPSCs: a novel approach to study NaV1.5 variants in a human cardiomyocyte environment

Figures

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  Figure 1. CRISPR-Cas9 genome editing to target SCN5a. (A) A schematic representation of the SCN5a locus and the sgRNA target sites located upstream and downstream from exon 6a. The sgRNA pair (blue) were designed to excise exon 6a in the SCN5a locus following CRISPR-Cas9 editing. The PCR primers used to screen and verify the nucleofected iPSC clones are shown in green. (B) PCR analysis of the positive iPSC clone used in this study amplified with primers shown in (A). Positive clone carrying a 685-bp PCR amplicon and non-nucleofected iPSC carrying a 962-bp amplicon. (C) PCR analysis on NaV1.5 cDNA using RNA extracted form iPSC-CM. The primers targeted the regions corresponding to exon 5 and exon 7 of NaV1.5 cDNA. PCR amplicons with a 92-bp deletion (NaV1.5 KO) or no deletion (control iPSC) corresponding to exon 6 are shown. (D) Sequencing chromatogram of the deleted PCR product in (C) from the NaV1.5 KO cell line showing the 92 bp deletion of exon 6 and the creation of stop codon induced by the frame shift of the reading frame. (E) Western blot analysis of NaV1.5 KO and control iPSC-CM. Anti-NaV1.5 (left panel) and the Anti-Pan NaV antibodies (right panel) were used to assess the expression…

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  Figure 2. Immunofluorescence staining of cardiac markers in NaV1.5 KO iPSC-CMs. NaV1.5 KO iPSC-CMs with organized contractile proteins, including myosin light chain 2v (MLC2v, green staining) and cardiac troponin T (cTnT, red staining). The bottom panels show merged images. Scale bar: 20 µm. Images were acquired using a Zeiss ImagesM2 LSM confocal microscope and were processed using ZEN software (Zeiss). This figure was made using IMAGEJ 1.52i (Java 1.8.0_66, http://imagej.nih.gov/ij).

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  Figure 3. Electrophysiological characterization of NaV1.5 KO iPSC-CMs. (A) Representative Na+ currents recorded in control and NaV1.5 KO iPSC-CMs with 35 or 140 mM NaCl in the external solution. The dashed line represents zero current. The currents were obtained using 200 ms pulses from − 100 to + 40 mV in + 5 mV increment. (B) Superposed train of APs from control and NaV1.5 KO at 1 Hz. Vertical bars at the bottom of the traces indicate the 3 ms stimulation pulse. (C) Close-up view of one AP showing the increase in the APD for NaV1.5 KO. (D) Box and whiskers summarizing the median (with quartiles and min to max values) of APD at 20 %, 50 %, or 90 % of repolarization at 1 Hz. *p < 0.05, **p < 0.01, ***p < 0.001 as determined using a nonparametric Mann–Whitney test. (E) Close-up view on the rising phase of representative APs traces. (F,G) Bar graphs showing the mean ± SEM of overshoot (F) and the maximal upstroke velocity (dV/dtmax) (G) of APs recorded at 1 Hz. ***p < 0.001 as determined using an unpaired t-test. Control n = 33 versus NaV1.5 KO n = 24. This figure was made using MICROSOFT POWERPOINT Version 2106 (build 14131.20278 Office, https://docs.microsoft.com/en-us/) and PRISM 8…

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  Figure 4. Slower conduction velocity and altered OAP parameters in NaV1.5 KO iPSC-CMs. (A) Representative optical action potentials (OAPs) recorded in control and NaV1.5 KO iPSC-CMs monolayers using di-4-ANEPPS dye. Blue arrows at the beginning and at the end of the pulse train indicate the cumulative delay of OAPs activation in NaV1.5 KO recording. (B) Representative activation maps at a pacing of 1 Hz. The symbol indicating the position of stimulating electrodes, and □ showing the position of the representative recordings. (C) Bar graphs summarizing the CVs, (D) the rise time, (E,F) action potential durations at 50 % or 80 % of repolarization and (G) the normalized amplitude of OAPs measured at stimulation frequencies of 0.5, 1 and 1.5 Hz in control (n = 9) and NaV1.5 KO (n = 7–9) iPSC-CM monolayers. Bars indicate SEM. **p < 0.01, ***p < 0.001 (control vs. NaV1.5 KO) as determined using unpaired t-test. A replicate (n) represents a single monolayer of iPSC-CMs on which optical mapping was performed. This figure was made using MICROSOFT POWERPOINT Version 2106 (build 14131.20278 Office, https://docs.microsoft.com/en-us/) and PRISM 8 Version 8.0.2 (263, https://www.graphpad.com/).

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  Figure 5. Altered calcium handling in NaV1.5 KO iPSC-CMs monolayers. (A) Representative Ca2+ transient traces of control and NaV1.5 KO iPSC-CMs monolayers. Blue arrows indicate the Ca2+ activation delay in NaV1.5 KO recording. (B) Representative Ca2+ activation maps at pacing of 1 Hz. The symbol indicating the position of stimulating electrodes, and □ showing the position of the representative recordings. (C) Bar graphs summarizing the Ca2+ propagation velocities (CaPVs), (D) the normalized amplitude. (E,F) the Ca2+ transient durations at 50 and 80% of the repolarization phase (TD50 and TD80), (G) the half time to peak, and (H) the time constant of repolarization (Decay tau) measured at stimulation frequencies of 0.5, 1 and 1.5 Hz in control (n = 12–15) and NaV1.5 KO (n = 6–9). (I) Bar graphs showing the spontaneous Ca2+ waves frequencies in control (n = 15) and NaV1.5 KO (n = 10) iPSC-CM monolayers. A replicate (n) represents a single monolayer of iPSC-CMs on which the optical mapping was performed. Bars indicate SEM. *p < 0.05, **p < 0.001, ***p < 0.001 as determined by unpaired t-test. This figure was made using MICROSOFT POWERPOINT Version 2106 (build 14131.20278 Office,…

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  Figure 6. Altered biophysical properties of Na+ channel in KO iPSC-CMs expressing NaV1.5/ΔQKP. (A) Representative Na+ currents recorded in control and KO iPSC-CMs transfected with NaV1.5/WT or NaV1.5/delQKP. The dashed line represents zero current. The currents were obtained using 200 ms pulses from − 100 to + 40 mV in + 5 mV increment. (B) Normalized intensity/voltage relationships (I/V) recorded in control (n = 14), NaV1.5/WT (n = 13) and NaV1.5/delQKP (n = 9) iPSC-CMs. Na+ current densities were measured by normalizing current amplitudes to membrane capacitance. Inset shows graph of Imax. (C) Steady-state activation of Na+ currents. Activation curves were generated using a standard Boltzmann distribution: G(V)/Gmax = 1/(1 + exp (− (V − V1/2)/k)). Inset shows graph of V1/2. (D) Steady-state inactivation of control (n = 14), NaV1.5/WT (n = 13) and NaV1.5/delQKP (n = 9). Inactivation 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. The inactivation values were fitted to a standard Boltzmann equation: I(V)/Imax = 1/(1 + exp ((V − V1/2)/k)) + C. Inset shows graph of V1/2 and k slope (E) Recovery from inactivation…

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  Figure 7. APD prolongation, dV/dt affected and arrhythmogenic events in KO iPSC-CMs expressing NaV1.5/delQKP. (A) Representative APs traces at a stimulation frequency of 1 Hz recorded in control and KO iPSC-CMs transfected with NaV1.5/WT or NaV1.5/delQKP. (B) Box and whiskers summarizing the median (with quartiles and min to max values) of APD at 20%, 50%, or 90% of repolarization at 1 Hz. *p < 0.05, ***p < 0.001: control versus NaV1.5/WT; #p < 0.05, ###p < 0.001: NaV1.5/WT versus NaV1.5/delQKP. A nonparametric Kruskal–Wallis test was performed. (C,D) Bar graph showing the mean ± SEM of the dV/dtmax (C) and the overshoot of APs (D) recorded at 1 Hz. ***p < 0.001: control versus NaV1.5/WT; ###p < 0.001: NaV1.5/WT versus NaV1.5/delQKP. Significance was determined by ANOVA and Dunnett’s post hoc tests. (E) Examples of DAD (red arrows) recorded at a stimulation of 0.5 Hz and EAD (red arrows) recorded in spontaneous AP (gap-free mode) in NaV1.5/delQKP. Control n = 33, NaV1.5/WT n = 22 and NaV1.5/delQKP n = 32. This figure was made using MICROSOFT POWERPOINT Version 2106 (build 14131.20278 Office, https://docs.microsoft.com/en-us/) and PRISM 8 Version 8.0.2 (263, https://www.graphpad.com/).