Vinculin variant M94I identified in sudden unexplained nocturnal death syndrome decreases cardiac sodium current

Figures

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  Figure 1. Genotyping of SUNDS victim. The heterozygous VCL variant M94I with a nucleotide change of G-A in the position 282 was identified in a SUNDS victim.

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  Figure 2. Cardiac histological views. No myocardial hypertrophy, disarray, significant infiltration of inflammatory cells, and substitution of fibrous/adipose tissue in the myocardium were observed in both SUNDS (A,C) and control (B,D). (A,B,C and D) were stained with H&E, Masson’s trichrome, respectively (all ×400).

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  Figure 3. Co-localization of VCL and SCN5A in human ventricular tissue slice. (A,B) Immunofluorescence staining of SCN5A (green), VCL (red) and overlay channel from control and case (SUNDS patient, VCL–M94I variant carrier) human ventricular tissue slices. The second row shows a higher magnification view of the area outlined in yellow the first row. The yellow color from overlay panel indicates co-localization of VCL and SCN5A. (C and D) Signal intensity profile of signals from selected areas (outlined with yellow dashed rectangle in second row of (A and B) were calculated and plotted from control and case slices respectively. Integrity of the fluorescence signals of both SCN5A (blue line) and VCL (red line) along transverse direction was aligned and plotted.

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  Figure 4. VCL directly interacts with SCN5A. (A) Mouse liver and heart (HT) tissue lysates were immunoprecipitated (IP) using VCL or SCN5A antibody and analyzed by Western blotting using the indicated antibodies; (B) Wild-type VCL (VCL-WT) and SCN5A were transfected into HEK293 cells for 24 h, cell lysates were IP using VCL or SCN5A antibody and analyzed by Western blotting using the indicated antibodies; (C) Mutant VCL (VCL-M94I) and SCN5A were transfected into HEK293 cells for 24 h, cell lysates were IP using VCL or SCN5A antibody and analyzed by Western blotting using the indicated antibodies; (D) VCL-WT, VCL-M94I and SCN5A were transfected into HEK293 cells for 24 h, cell lysates were IP using VCL or SCN5A antibody and analyzed by Western blotting using the indicated antibodies. The results are representative of three independent experiments.

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  Figure 5. Electrophysiological properties of cardiac sodium channel in HEK293 cells co-expressing SCN5A and either WT or mutant VCL. (A) Representative whole-cell current traces showing peak INa under both normal (pH 7.4) and moderate acidosis (pH 7.0) condition in HEK293 cells expressing SCN5A and either WT or mutant VCL. (B) Summary data of peak INa densities from every group. The number of tested cells is indicated above the bar. *p < 0.05, **p < 0.01.

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  Figure 6. Voltage–dependent gating for SCN5A co-expressed with VCL in HEK293 cells. (A) Under normal pH condition, M94I caused a statistically significant depolarizing shift in activation of cardiac sodium channel by 2.7 mV compared to WT. Vrev = 84.8 mV. (B) Under pH 7.0, M94I showed a significant repolarizing shift by 4.1 mV in inactivation of cardiac sodium channel compared with WT at pH 7.4.

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  Figure 7. Electrophysiological properties of cardiac sodium channel in iPSC-CMs expressing either WT or mutant VCL. (A) Representative whole-cell current traces showing peak INa under both normal (pH 7.4) and moderate acidosis (pH 7.0) condition in iPSCs-CM expressing either WT or mutant VCL. (B) Summary data of peak INa densities from every group. The number of tested cells is indicated above the bar. *p < 0.05.

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  Figure 8. Voltage–dependent gating for cardiac sodium channel in iPSC-CMs expressing with VCL. (A) Between each group, no statistically significant difference in activation of cardiac sodium channel was observed. Vrev = 63.2 mV. (B) Under pH 7.0, M94I caused a significant repolarizing shift by 15.3 mV in inactivation of cardiac sodium channel compared to WT at pH 7.4.