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

H2O2 reduces NaV1.5 expression.Western blot analysis showed that NaV1.5 protein was significantly decreased (*p<0.05) in HL-1 cells treated with 25 µM H2O2 for 48 h hours (n = 3) compared with the control group (n = 3) (A, C). RT-PCR analysis showed that NaV1.5 mRNA significantly reduced (p*<0.05) in HL-1 cells treated with 25 µM H2O2 for 48 hours (n = 3) compared with the control group (n = 4) (B, D).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3293505&req=5

pone-0032738-g007: H2O2 reduces NaV1.5 expression.Western blot analysis showed that NaV1.5 protein was significantly decreased (*p<0.05) in HL-1 cells treated with 25 µM H2O2 for 48 h hours (n = 3) compared with the control group (n = 3) (A, C). RT-PCR analysis showed that NaV1.5 mRNA significantly reduced (p*<0.05) in HL-1 cells treated with 25 µM H2O2 for 48 hours (n = 3) compared with the control group (n = 4) (B, D).

Mentions: H2O2 inhibiting NaV1.5 expression is through the Foxo1 signaling pathway: Increase of Foxo1 nuclear localization by H2O2 is expected to reduce NaV1.5 expression. In order to confirm this assumption, RNA and protein were extracted from HL-1 cells treated with 25 µM H2O2 for 48 hours. Analyses of RT-PCR products and Western blot results showed that both NaV1.5 protein and mRNA levels were significantly decreased (p<0.05) (Fig. 7A, C and B, D) in comparison with controls. In order to determine if H2O2 reducing NaV1.5 expression was through the Foxo1 signaling, 100 MOI Adv-scramble RNAi and 100 MOI Adv-Foxo1-RNAi were used to infect HL-1 cells and 25 µM H2O2 was added in the culture medium after the cells expressed scramble RNAi and Foxo1-RNAi for 24 hours. Both NaV1.5 mRNA and protein levels were significantly increased (p<0.05 and p<0.01, respectively) in the cells expressing Foxo1 RNAi with a 48-hour H2O2 treatment when compared with the cells expressing scramble RNAi with 48 hour H2O2 treatment (Fig. 8A, C and B, D).


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)

H2O2 reduces NaV1.5 expression.Western blot analysis showed that NaV1.5 protein was significantly decreased (*p<0.05) in HL-1 cells treated with 25 µM H2O2 for 48 h hours (n = 3) compared with the control group (n = 3) (A, C). RT-PCR analysis showed that NaV1.5 mRNA significantly reduced (p*<0.05) in HL-1 cells treated with 25 µM H2O2 for 48 hours (n = 3) compared with the control group (n = 4) (B, D).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0032738-g007: H2O2 reduces NaV1.5 expression.Western blot analysis showed that NaV1.5 protein was significantly decreased (*p<0.05) in HL-1 cells treated with 25 µM H2O2 for 48 h hours (n = 3) compared with the control group (n = 3) (A, C). RT-PCR analysis showed that NaV1.5 mRNA significantly reduced (p*<0.05) in HL-1 cells treated with 25 µM H2O2 for 48 hours (n = 3) compared with the control group (n = 4) (B, D).
Mentions: H2O2 inhibiting NaV1.5 expression is through the Foxo1 signaling pathway: Increase of Foxo1 nuclear localization by H2O2 is expected to reduce NaV1.5 expression. In order to confirm this assumption, RNA and protein were extracted from HL-1 cells treated with 25 µM H2O2 for 48 hours. Analyses of RT-PCR products and Western blot results showed that both NaV1.5 protein and mRNA levels were significantly decreased (p<0.05) (Fig. 7A, C and B, D) in comparison with controls. In order to determine if H2O2 reducing NaV1.5 expression was through the Foxo1 signaling, 100 MOI Adv-scramble RNAi and 100 MOI Adv-Foxo1-RNAi were used to infect HL-1 cells and 25 µM H2O2 was added in the culture medium after the cells expressed scramble RNAi and Foxo1-RNAi for 24 hours. Both NaV1.5 mRNA and protein levels were significantly increased (p<0.05 and p<0.01, respectively) in the cells expressing Foxo1 RNAi with a 48-hour H2O2 treatment when compared with the cells expressing scramble RNAi with 48 hour H2O2 treatment (Fig. 8A, C and B, D).

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