Limits...
Reactive oxygen species suppress cardiac NaV1.5 expression through Foxo1.

Mao W, You T, Ye B, Li X, Dong HH, Hill JA, Li F, Xu H - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York, USA.

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.

Show MeSH

Related in: MedlinePlus

Foxo1 inhibits Na+ channel activity.Foxo1-AA-GFP was localized in HL-1 cardiomyocyte nuclei (B) while GFP was seen in both nuclei and cytoplasm (A). Whole cell Na+ currents were recorded during 80 ms depolarizing voltage steps to potentials between −60 and +60 mV from a holding potential of −100 mV in HL-1 cells expressing GFP or Foxo1-AA-GFP for 36 hours. The typical whole cell recording traces showed that robust Na+ currents were present in HL-1 cells expressing GFP (C) while remarkable reduction of Na+ currents in these cells expressing Foxo1-AA-GFP (D).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3293505&req=5

pone-0032738-g005: Foxo1 inhibits Na+ channel activity.Foxo1-AA-GFP was localized in HL-1 cardiomyocyte nuclei (B) while GFP was seen in both nuclei and cytoplasm (A). Whole cell Na+ currents were recorded during 80 ms depolarizing voltage steps to potentials between −60 and +60 mV from a holding potential of −100 mV in HL-1 cells expressing GFP or Foxo1-AA-GFP for 36 hours. The typical whole cell recording traces showed that robust Na+ currents were present in HL-1 cells expressing GFP (C) while remarkable reduction of Na+ currents in these cells expressing Foxo1-AA-GFP (D).

Mentions: To test the hypothesis that activation of Foxo1 reducing NaV1.5 expression decreases functional Na+ currents, whole-cell voltage-clamp Na+ current recordings were performed on HL-1 cells infected with 50 MOI Adv-CMV-GFP and 50 MOI Adv-CMV-Foxo1-AA-GFP for 48 hours, respectively. Foxo1-AA-GFP was localized in the nuclei (Fig. 5B), while GFP was seen in both cytoplasm and nuclei (Fig. 5A). Recording traces of rapidly activating and inactivating Na+ currents from GFP and Foxo1-AA-GFP-expressing cells, respectively, 36 hours after adenovirus infection, are illustrated in Figure 5C and D. Na+ currents were significantly inhibited by expression of Foxo1-AA-GFP, and peak Na+ current density was significantly lower (p<0.01) in cells expressing Foxo1-AA-GFP (19.6±5.6 pA/pF, at −25 mV) than in cells expressing GFP (67.9±9.6 pA/pF, at −25 mV).


Reactive oxygen species suppress cardiac NaV1.5 expression through Foxo1.

Mao W, You T, Ye B, Li X, Dong HH, Hill JA, Li F, Xu H - PLoS ONE (2012)

Foxo1 inhibits Na+ channel activity.Foxo1-AA-GFP was localized in HL-1 cardiomyocyte nuclei (B) while GFP was seen in both nuclei and cytoplasm (A). Whole cell Na+ currents were recorded during 80 ms depolarizing voltage steps to potentials between −60 and +60 mV from a holding potential of −100 mV in HL-1 cells expressing GFP or Foxo1-AA-GFP for 36 hours. The typical whole cell recording traces showed that robust Na+ currents were present in HL-1 cells expressing GFP (C) while remarkable reduction of Na+ currents in these cells expressing Foxo1-AA-GFP (D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293505&req=5

pone-0032738-g005: Foxo1 inhibits Na+ channel activity.Foxo1-AA-GFP was localized in HL-1 cardiomyocyte nuclei (B) while GFP was seen in both nuclei and cytoplasm (A). Whole cell Na+ currents were recorded during 80 ms depolarizing voltage steps to potentials between −60 and +60 mV from a holding potential of −100 mV in HL-1 cells expressing GFP or Foxo1-AA-GFP for 36 hours. The typical whole cell recording traces showed that robust Na+ currents were present in HL-1 cells expressing GFP (C) while remarkable reduction of Na+ currents in these cells expressing Foxo1-AA-GFP (D).
Mentions: To test the hypothesis that activation of Foxo1 reducing NaV1.5 expression decreases functional Na+ currents, whole-cell voltage-clamp Na+ current recordings were performed on HL-1 cells infected with 50 MOI Adv-CMV-GFP and 50 MOI Adv-CMV-Foxo1-AA-GFP for 48 hours, respectively. Foxo1-AA-GFP was localized in the nuclei (Fig. 5B), while GFP was seen in both cytoplasm and nuclei (Fig. 5A). Recording traces of rapidly activating and inactivating Na+ currents from GFP and Foxo1-AA-GFP-expressing cells, respectively, 36 hours after adenovirus infection, are illustrated in Figure 5C and D. Na+ currents were significantly inhibited by expression of Foxo1-AA-GFP, and peak Na+ current density was significantly lower (p<0.01) in cells expressing Foxo1-AA-GFP (19.6±5.6 pA/pF, at −25 mV) than in cells expressing GFP (67.9±9.6 pA/pF, at −25 mV).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York, USA.

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.

Show MeSH
Related in: MedlinePlus