Activation of Wnt/β-catenin signaling by hydrogen peroxide transcriptionally inhibits NaV1.5 expression

Oxidants and canonical Wnt/beta-catenin signaling have been shown to decrease cardiac Na+ channel activity by suppressing Na(V)1.5 expression. Our aims are to determine if hydrogen peroxide (H2O2), one oxidant of reactive oxygen species (ROS), activates Wnt/beta-catenin signaling and promotes beta-catenin nuclear activity, leading to suppression of Na(V)1.5 expression and if this suppression requires the interaction of beta-catenin with its nuclear partner, TCF4 (also called TCF7L2) to decrease SCN5a promoter activity. The results demonstrated that H2O2 increased beta-catenin, but not TCF4 nuclear localization determined by immunofluorescence without affecting total beta-catenin protein level. Furthermore, H2O2 exerted a dose- and time-dependent suppressive effect on Na(V)1.5 expression. RT-PCR and/or Western blot analyses revealed that overexpressing active form of beta-catenin or stabilizing beta-catenin by GSK-3 beta inhibitors, LiCl and Bio, suppressed Na(V)1.5 expression in HL-1 cells. In contrast, destabilization of beta-catenin by a constitutively active GSK-3 beta mutant (S9A) upregulated Na(V)1.5 expression. Whole-cell recording showed that LiCl significantly inhibited Na+ channel activity in these cells. Using immunoprecipitation (IP), we showed that beta-catenin interacted with TCF4 indicating that beta-catenin as a co-transfactor, regulates Na(V)1.5 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 beta-catenin and TCF4 were recruited in the SCN5a promoter, and regulated its activity. Luciferase promoter assays exhibited that beta-catenin inhibited the SCN5a promoter activity at a dose-dependent manner and this inhibition required TCF4. Small interfering (Si) RNA targeting beta-catenin significantly increased SCN5a promoter activity, leading to enhanced Na(V)1.5 expression. As expected, beta-catenin SiRNA prevents H2O2 suppressive effects on both SCN5a promoter activity and Na(V)1.5 expression. Our findings indicate that H2O2 inhibits Na(V)1.5 expression by activating the Wnt/beta-catenin signaling and beta-catenin interacts with TCF4 to transcriptionally suppress cardiac Na(V)1.5 expression. (C) 2016 Elsevier Inc. All rights reserved.