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Suppression of the cardiac sodium channel NaV1.5 leads to fatal arrhythmias in ischemic heart disease (IHD). However, the transcriptional regulation of NaV1.5 in cardiac ischemia is still unclear. Our studies are aimed to investigate the expression of enhancer of zeste homolog 2 (EZH2) in IHD and regulation of cardiac NaV1.5 expression by EZH2. Human heart tissue was obtained from IHD and non-failing heart (NFH) patients; mouse heart tissue was obtained from the peri-infarct zone of hearts with myocardial infarction (MI) and hearts with a sham procedure. Protein and mRNA expression were measured by immunoblotting, immunostaining, and qRT-PCR. Protein-DNA binding and promoter activity were analyzed by ChIP-qPCR and luciferase assays, respectively. Na+ channel activity was assessed by whole-cell patch clamp recordings. EZH2 and H3K27me3 were increased while NaV1.5 expression was reduced in IHD hearts and in mouse MI hearts compared to the controls. Reduced NaV1.5 and increased EZH2 mRNA levels were observed in mouse MI hearts. A selective EZH2 inhibitor, GSK126 decreased H3K27me3 and elevated NaV1.5 in HL-1 cells. Silencing of EZH2 expression decreased H3K27me3 and increased NaV1.5 in these cells. EZH2 and H3K27me3 were enriched in the promoter regions of Scn5a and were decreased by treatment with EZH2 siRNA. GSK126 inhibited the enrichment of H3K27me3 in the Scn5a promoter and enhanced Scn5a transcriptional activity. GSK126 significantly increased Na+ channel activity. Taken together, EZH2 is increased in ischemic hearts and epigenetically suppresses Scn5a transcription by H3K27me3, leading to decreased NaV1.5 expression and Na+ channel activity underlying the pathogenesis of arrhythmias.

Increased nuclear β-catenin interacting with T-cell factor 4 (TCF4) affects the expression of target genes including SCN5A in ischemic heart disease, which is characterized by frequent ventricular tachycardia/fibrillation. A complex of β-catenin and TCF4 inhibits cardiac Na+ channel activity by reducing NaV1.5 expression through suppressing SCN5A promoter activity in HL-1 cardiomyocytes. LF3, a 4-thioureido-benzenesulfonamide derivative and an inhibitor of β-catenin/TCF4 interaction, has been shown to block the self-renewal capacity of cancer stem cells. We performed studies to determine if LF3 can reverse suppressive effects of β-catenin/TCF4 signaling on the expression of NaV1.5 in HL-1 cardiomyocytes. Western blotting and real-time qRT-PCR analyses showed that 10 μM LF3 significantly increased the expression of NaV1.5 but it did not alter β-catenin and TCF4 expression. Subcellular fractionation analysis demonstrated that LF3 significantly increased the levels of NaV1.5 in both membrane and cytoplasm. Whole-cell patch-clamp recordings revealed that Na+ currents were significantly increased with no changes in the steady-state parameters, activation and inactivation time constants and recovery from inactivation of Na+ channel in HL-1 cells treated with LF3. Immunoprecipitation exhibited that LF3 blocked the interaction of β-catenin and TCF4. Luciferase reporter assays performed in HEK 293 cells and HL-1 revealed that LF3 increased the SCN5A promoter activity in HL-1 cells and prevented β-catenin suppressive effect on SCN5A promoter activity in HEK 293 cells. Taken together, we conclude that LF3, an inhibitor of β-catenin/TCF4 interaction, elevates NaV1.5 expression, leading to increase Na+ channel activity in HL-1 cardiomyocytes.

Increased nuclear β-catenin interacting with T-cell factor 4 (TCF4) affects the expression of target genes including SCN5A in ischemic heart disease, which is characterized by frequent ventricular tachycardia/fibrillation. A complex of β-catenin and TCF4 inhibits cardiac Na(+) channel activity by reducing Na(V)1.5 expression through suppressing SCN5A promoter activity in HL-1 cardiomyocytes. LF3, a 4-thioureido-benzenesulfonamide derivative and an inhibitor of β-catenin/TCF4 interaction, has been shown to block the self-renewal capacity of cancer stem cells. We performed studies to determine if LF3 can reverse suppressive effects of β-catenin/TCF4 signaling on the expression of Na(V)1.5 in HL-1 cardiomyocytes. Western blotting and real-time qRT-PCR analyses showed that 10 μM LF3 significantly increased the expression of Na(V)1.5 but it did not alter β-catenin and TCF4 expression. Subcellular fractionation analysis demonstrated that LF3 significantly increased the levels of Na(V)1.5 in both membrane and cytoplasm. Whole-cell patch-clamp recordings revealed that Na(+) currents were significantly increased with no changes in the steady-state parameters, activation and inactivation time constants and recovery from inactivation of Na(+) channel in HL-1 cells treated with LF3. Immunoprecipitation exhibited that LF3 blocked the interaction of β-catenin and TCF4. Luciferase reporter assays performed in HEK 293T cells and HL-1 revealed that LF3 increased the SCN5A promoter activity in HL-1 cells and prevented β-catenin suppressive effect on SCN5A promoter activity in HEK 293 cells. Taken together, we conclude that LF3, an inhibitor of β-catenin/TCF4 interaction, elevates Na(V)1.5 expression, leading to increase Na(+) channel activity in HL-1 cardiomyocytes.

Aims Cardiomyocyte (CM) proliferation increases from the inner trabecular to outer compact myocardium in fetal hearts. We determined if canonical Wnt signaling has directional and graded activity to maintain this CM proliferation gradient. Moreover, we investigated whether perturbation of Wnt signaling intensity could modulate CM proliferative activity. Methods and results With confocal microscopy and image analysis we found that the Wnt effector, β-catenin, formed a signaling gradient which positively correlated with CM proliferative activity across ventricular walls of wild type (WT) embryos at embryonic day (E) 13.5 and 17.5. Negative Wnt regulators, adenomatosis polyposis coli (APC), had a reverse distribution pattern. The activation of canonical Wnt/β-catenin signaling by deletion of Apc in CMs led to ventricular hyperplasia with no adverse effects on fetal survival or CM differentiation. In contrast, cardiac deletion of β-catenin resulted in ventricular hypoplasia and fetal demise by E14.5. We further revealed differential distribution and regulation of three cyclin Ds in fetal hearts. Cyclin D1 was mainly expressed in endothelial cells. Although both cyclin D2 and D3 were present in CMs, only cyclin D2 was regulated by Wnt signaling perturbation: downregulation by β-catenin deletion and upregulation by Apc knockout. Conclusion Canonical Wnt signaling is asymmetrical and graded across ventricular walls and positively regulates CM proliferation via cyclin D2.

Our in vitro studies revealed that a transcription factor, Forkhead box protein O1 (FoxO1), negatively regulates the expression of NaV1.5, a main α subunit of the cardiac Na(+) channel, by altering the promoter activity of SCN5a in HL-1 cardiomyocytes. The in vivo role of FoxO1 in the regulation of cardiac NaV1.5 expression remains unknown. The present study aimed to define the role of FoxO1 in the regulation of NaV1.5 expression and cardiac Na(+) channel activity in mouse ventricular cardiomyocytes and assess the cardiac electrophysiological phenotype of mice with cardiac FoxO1 deletion. Tamoxifen-induced and cardiac-specific FoxO1 deletion was confirmed by polymerase chain reaction (PCR). Cardiac FoxO1 deletion failed to result in either cardiac functional changes or hypertrophy as assessed by echocardiography and individual ventricular cell capacitances, respectively. Western blotting showed that FoxO1 was significantly decreased while NaV1.5 protein level was significantly increased in mouse hearts with FoxO1 deletion. Reverse transcription-PCR (RT-PCR) revealed that FoxO1 deletion led to an increase in NaV1.5 and Na(+) channel subunit β3 mRNA, but not β1, 2, and 4, or connexin 43. Whole patch-clamp recordings demonstrated that cardiac Na(+) currents were significantly augmented by FoxO1 deletion without affecting the steady-state activation and inactivation, leading to accelerated depolarization of action potentials in mouse ventricular cardiomyocytes. Electrocardiogram recordings showed that the QRS complex was significantly shortened and the P wave amplitude was significantly increased in conscious and unrestrained mice with cardiac FoxO1 deletion. NaV1.5 expression was decreased in the peri-infarct (border-zone) of mice with myocardial infarction and FoxO1 accumulated in the cardiomyocyte nuclei of chronic ischemic human hearts. Our findings indicate that FoxO1 plays an important role in the regulation of NaV1.5 and β3 subunit expressions as well as Na(+) channel activity in the heart and that FoxO1 is involved in the modulation of NaV1.5 expression in ischemic heart disease.