title: Reactive oxygen species suppress cardiac NaV1.5 expression through Foxo1.
abstract: Na(V)1.5 is a cardiac voltage-gated Na(+) channel αsubunit and is encoded by the SCN5a gene. The activity of this channel determines cardiac depolarization and electrical conduction. Channel defects, including mutations and decrease of channel protein levels, have been linked to the development of cardiac arrhythmias. The molecular mechanisms underlying the regulation of Na(V)1.5 expression are largely unknown. Forkhead box O (Foxo) proteins are transcriptional factors that bind the consensus DNA sequences in their target gene promoters and regulate the expression of these genes. Comparative analysis revealed conserved DNA sequences, 5'-CAAAACA-3' (insulin responsive element, IRE), in rat, mouse and human SCN5a promoters with the latter two containing two overlapping Foxo protein binding IREs, 5'-CAAAACAAAACA-3'. This finding led us to hypothesize that Foxo1 regulates Na(V)1.5 expression by directly binding the SCN5a promoter and affecting its transcriptional activity. In the present study, we determined whether Foxo1 regulates Na(V)1.5 expression at the transcriptional level and also defined the role of Foxo1 in hydrogen peroxide (H(2)O(2))-mediated Na(V)1.5 suppression in HL-1 cardiomyocytes using chromatin immunoprecipitation (ChIP), constitutively nuclear Foxo1 expression, and RNAi Foxo1 knockdown as well as whole cell voltage-clamp recordings. ChIP with anti-Foxo1 antibody and follow-up semi-quantitative PCR with primers flanking Foxo1 binding sites in the proximal SCN5a promoter region clearly demonstrated enrichment of DNA, confirming Foxo1 recruitment to this consensus sequence. Foxo1 mutant (T24A/S319A-GFP, Foxo1-AA-GFP) was retained in nuclei, leading to a decrease of Na(V)1.5 expression and Na(+) current, while silencing of Foxo1 expression by RNAi resulted in the augmentation of Na(V)1.5 expression. H(2)O(2) significantly reduced Na(V)1.5 expression by promoting Foxo1 nuclear localization and this reduction was prevented by RNAi silencing Foxo1 expression. These studies indicate that Foxo1 negatively regulates Na(V)1.5 expression in cardiomyocytes and reactive oxygen species suppress Na(V)1.5 expression through Foxo1.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York, USA.
authors.forename: Weike
authors.initials: W
authors.lastname: Mao
authors.forename: Tao
authors.initials: T
authors.lastname: You
authors.forename: Bo
authors.initials: B
authors.lastname: Ye
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.forename: Henry H
authors.initials: HH
authors.lastname: Dong
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Hill
authors.forename: Faqian
authors.initials: F
authors.lastname: Li
authors.forename: Haodong
authors.initials: H
authors.lastname: Xu
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D016877:Oxidants / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D034622:RNA Interference; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D062554:NAV1.5 Voltage-Gated Sodium Channel; D016877:Oxidants; D017382:Reactive Oxygen Species; C568320:SCN5A protein, human; C568321:Scn5a protein, mouse; C568322:Scn5a protein, rat; D015222:Sodium Channels; D006861:Hydrogen Peroxide