Activation of Wnt/β-catenin signaling by hydrogen peroxide transcriptionally inhibits Na_V1.5 expression

Oxidants and canonical Wnt/β-catenin signaling have been shown to decrease cardiac Na⁺ channel activity by suppressing Na_V1.5 expression. Our aims are to determine if hydrogen peroxide (H₂O₂), one oxidant of reactive oxygen species (ROS), activates Wnt/β-catenin signaling and promotes β-catenin nuclear activity, leading to suppression of Na_V1.5 expression and if this suppression requires the interaction of β-catenin with its nuclear partner, TCF4 (also called TCF7L2) to decrease SCN5a promoter activity. The results demonstrated that H₂O₂ increased β-catenin, but not TCF4 nuclear localization determined by immunofluorescence without affecting total β-catenin protein level. Furthermore, H₂O₂ exerted a dose- and time-dependent suppressive effect on Na_V1.5 expression. RT-PCR and/or Western blot analyses revealed that overexpressing active form of β-catenin or stabilizing β-catenin by GSK-3β inhibitors, LiCl and Bio, suppressed Na_V1.5 expression in HL-1 cells. In contrast, destabilization of β-catenin by a constitutively active GSK-3β mutant (S9A) upregulated Na_V1.5 expression. Whole-cell recording showed that LiCl significantly inhibited Na⁺ channel activity in these cells. Using immunoprecipitation (IP), we showed that β-catenin interacted with TCF4 indicating that β-catenin as a co-transfactor, regulates Na_V1.5 expression through TCF4. Analyses of the SCN5a promoter sequences among different species by using VISTA tools indicated that SCN5a promoter harbors TCF4 binding sites. Chromatin IP assays demonstrated that both β-catenin and TCF4 were recruited in the SCN5a promoter, and regulated its activity. Luciferase promoter assays exhibited that β-catenin inhibited the SCN5a promoter activity at a dose-dependent manner and this inhibition required TCF4. Small interfering (Si) RNA targeting β-catenin significantly increased SCN5a promoter activity, leading to enhanced Na_V1.5 expression. As expected, β-catenin SiRNA prevents H₂O₂ suppressive effects on both SCN5a promoter activity and Na_V1.5 expression. Our findings indicate that H₂O₂ inhibits Na_V1.5 expression by activating the Wnt/β-catenin signaling and β-catenin interacts with TCF4 to transcriptionally suppress cardiac Na_V1.5 expression.