An East Asian Common Variant Vinculin P.Asp841His Was Associated With Sudden Unexplained Nocturnal Death Syndrome in the Chinese Han Population

Figures

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  Figure 1. Genotyping of sudden unexplained nocturnal death syndrome (SUNDS) victim. The common variant, Asp841His, of the vinculin (VCL) gene is confirmed by Sanger sequencing in 6 SUNDS cases with heterozygous G/C and in 2 SUNDS cases with homozygosis C/C at nucleotide 841. An arrow shows the position of the mutation. The Asp841His substitution involves an amino acid that is highly conserved among species. VCL accession numbers: human, NP_054706.1; chimpanzee, XP_507854; mouse, NP_033528.3; rat, NP_001100718.1; cow, NP_001178299.1; chicken, NP_990772.1; zebrafish, NP_001122153.1.

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  Figure 2. Colocalization of vinculin (VCL) and cardiac sodium channel (SCN5A) in human ventricular tissue slice. A and B, Immunofluorescent staining of SCN5A (green), VCL (red), and overlay channel from control and case (a homozygous D841H carrier) human ventricular tissue sections, presented from a normal and magnification field of view, respectively. Magnification field of view were labeled by yellow solid boxes in the panel. Yellow color from overlay panel indicates the colocalization of VCL and SCN5A. C and D, Signal intensity profile of signals from selected areas (shown in the panel) were calculated and plotted from control and case slices, respectively. The selected areas were labeled by yellow dashed boxes in the magnification in (A) and (B). Integrity of the fluorescence signals of both SCN5A (blue line) and VCL (red line) along transverse direction was aligned and plotted. A.U. indicates arbitrary units; ID, intercalated disk.

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  Figure 3. Vinculin (VCL) interacts with cardiac sodium channel (SCN5A) in vivo and in vitro. A, Heart tissue (HT) lysates from mouse were immunoprecipitated (IP) using SCN5A or VCL antibody and analyzed by western blotting using the indicated antibodies. Because of the negative expression of SCN5A, mouse liver tissue was used as a control. (B) SCN5A and wild‐type VCL (VCL‐WT) were transfected into HEK293 cells for 1 day, and cell lysates were IP and analyzed by western blotting. (C) VCL‐D841H and SCN5A were transfected into HEK293 cells for 1 day, and cell lysates were IP and analyzed by western blotting. (D) VCL‐WT, VCL‐D841H, and SCN5A were transfected into HEK293 cells for 1 day, and cell lysates were IP and analyzed by western blotting. IgG indicates immunoblobulin G; MW, molecular weight.

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  Figure 4. Electrophysiological properties of cardiac sodium channel (SCN5A) in HEK293 cells coexpressing SCN5A and either wild‐type vinculin (VCL‐WT) or VCL‐D841H. A, Representative whole‐cell current traces showing peak sodium current (IN a) under both normal (pH 7.4) and acidosis (pH 7.0) conditions in HEK293 cells expressing SCN5A and either WT or variant VCL‐D841H. B, Summary data of peak IN a densities from every group. The number of tested cells is indicated above the bar. *P<0.05.

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  Figure 5. Voltage‐dependent gating for cardiac sodium channel (SCN5A) coexpressed with vinculin (VCL) in HEK293 cells. A, Between each group, no significant difference in activation of SCN5A was observed. B, Under pH 7.0, D841H showed a significant repolarizing shift by 3.0 mV in inactivation of SCN5A compared to WT at pH 7.4.

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  Figure 6. Electrophysiological properties of cardiac sodium channels in induced pluripotent stem‐cell–derived cardiomyocytes (iPSC‐CMs) expressing either wild‐type vinculin (VCL‐WT) or VCL‐D841H. A, Representative whole‐cell current traces showing peak sodium current (IN a) under both normal (pH 7.4) and acidosis (pH 7.0) conditions in iPSC‐CMs expressing either WT or variant VCL‐D841H. B, Summary data of peak IN a densities from every group. The number of tested cells is indicated above the bar. *P<0.05.

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  Figure 7. Voltage‐dependent gating for cardiac sodium channel in induced pluripotent stem‐cell–derived cardiomyocytes expressing with vinculin (VCL). A, There was no statistically significant difference in activation of cardiac sodium channel between each group. B, Under pH 7.0 conditions, D841H caused a significant repolarizing shift by 25.6 mV in inactivation of cardiac sodium channel compared to wild type (WT) at pH 7.4.

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  Figure 8. Late sodium current characterized in HEK293 cells coexpressing cardiac sodium channel (SCN5A) and vinculin (VCL). Representative traces showing increased late sodium current (IN a) associated with SCN5A‐H558R+VCL‐D841H compared with SCN5A‐WT+VCL‐WT, or SCN5A‐WT+VCL‐D841H, or SCN5A‐H558R+VCL‐WT.