Cellular-level analyses of SCN5A mutations in left ventricular noncompaction cardiomyopathy suggest electrophysiological mechanisms for ventricular tachycardia

Figures

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  Fig. 1. Topologic location of SCN5A variants and sequence analysis of the constructed SCN5A variants A The α subunit of the SCN5A cardiac sodium channel is a transmembrane protein with four domains. The red points (•) represent seven heterozygous variants (p.G292S–SCN5A, p.H558R–SCN5A, p.P1090L-SCN5A, p.R1195H–SCN5A, p.R1193Q-SCN5A, p.V1951L-SCN5A, p.R1193Q-SCN5A and p.A1180V–SCN5A) located in intracellular loops, and the blue points (•) represent the heterozygous variant p.G292S–SCN5A located in extracellular loops. B Sequence analysis of the constructed SCN5A variants. C Electrocardiograms of a LVNC patient carrying the V1951L mutation.

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  Fig. 2. Location and expression of NaV1.5 in human cardiac tissues and hESC-CMs. A Immunohistochemistry was used to evaluate the distribution of the NaV1.5 protein in both normal human hearts and left ventricular noncompaction (LVNC) tissues. The localization of NaV1.5 in the T-tubule system and intercalated discs is indicated by black arrows in the representative images. The upper panels contain a box delineating the regions that are magnified in the lower panels. Scale bars measuring 100 μm and 25 μm are provided in the upper and lower panels, respectively. n > 3 for each group. B Images of immunofluorescence staining for actin (red), NaV1.5 (yellow), N-cadherin (red), and NaV1.5 (yellow) proteins (in the right panels) in hESC-CMs transfected with SCN5A variants or an empty virus.

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  Fig. 3. Analysis of the SCN5A variants in CHO–K1 cells. A Representative whole-cell current traces of WT and variant sodium channel currents. (Inset) The recording protocol. The experimental protocol consisted of applying different depolarizing potentials ranging from −80 mV to +15 mV, starting from a resting potential of −120 mV. The stimulus frequency used was 0.5 Hz to elicit the recorded currents. B Activation curves of the WT or variant channels. C Bar graphs showing the V1/2 values of the activation curves for the WT and variant strains. D Representative steady-state fast inactivation current traces of WT NaV1.5 channels and variants. E The steady-state fast inactivation curves exhibit a dependence on voltage. F V1/2 values summarized from panel E. G Voltage-dependent characteristics of the steady-state slow inactivation curves of variant or wild-type channels. H Recovery of WT and variant channels from inactivation. I Bar graph showing the recovery time constant τ (ms). The details are provided in Table S1.

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  Fig. 4. Effects of NaV1.5 WT and variant NaV1.5 on the contraction of hESC-CMs. A Representative traces of impedance detected by the CE96 system in the WT, variant and control groups. SCN5A variants induce a secondary beat and degenerate into fibrillation. #: secondary beat. B Bar graphs showing the beat rate for the WT and variant channel groups. C Bar graphs showing the beating amplitude for the WT and variant channel groups. D Bar graphs showing the pulse width (90 %) at which the WT and variant channel groups were beating. The data are shown as the mean ± SEM. The details of the beat rate, amplitude and pulse width are provided in Table S3. (***P < 0.001, **P < 0.01, *P < 0.05; one-way ANOVA).

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  Fig. 5. Effects of NaV1.5 WT and variant NaV1.5 on the extracellular field potential (FP) A Representative multielectrode array traces of the mean extracellular field potential detected by the CE96 system in the WT, variant and control groups.

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  Fig. 6. Effects of NaV1.5 WT and variant NaV1.5 on the frequency of field potentials in hESC-CMs. A Representative traces of extracellular field potential emission detected by the CE96 system in the WT, variant and control groups.

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  Fig. 7. Acute cardiac effects of Lidocaine, an INa ion channel modulator and antiarrhythmic drug on cell contraction in the variant-SCN5A group. A Cardiac contractility was assessed by examining changes in beat rate and amplitude. The expected acute effects of cardiac ion channel modulators of INa (Lidocaine,100 μM) were confirmed by the traces above showing spontaneous contractile activity under control conditions (pre-drug) and in the presence of the drug after 30 min of incubation. Lidocaine can slow the acceleration and fibrillation of hESC-CMs caused by SCN5A variants. B The scatter plot shows the effect of 100 μM Lidocaine on the beat rate of hESC-CMs. C The scatter plot illustrates the impact of a concentration of 100 μM Lidocaine on the beat amplitude of human embryonic stem cell-derived cardiomyocytes (hESC-CMs). #: secondary beat. The data are shown as the mean ± SEM. The details of the beat rate and amplitude are provided in Table S3.