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A small-molecule LF3 abrogates β-catenin/TCF4-mediated suppression of NaV1.5 expression in HL-1 cardiomyocytes
Limei Zhao1, Lihua Sun1, Yan Lu1
1Department of Pathology, Center for Cardiovascular Biology and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 90105, United States of America.
Abstract:
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.
Insights
LF3, a novel compound, reverses the suppressive effects of beta-catenin/TCF4 signaling on cardiac sodium channel NaV1.5 expression. This increases sodium channel activity in heart cells, potentially aiding treatment for arrhythmias.
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Pharmacology
Background:
- The beta-catenin/T-cell factor 4 (TCF4) complex suppresses SCN5A expression, reducing cardiac NaV1.5 levels and contributing to arrhythmias in ischemic heart disease.
- This interaction inhibits cardiac Na+ channel activity by decreasing NaV1.5 expression via SCN5A promoter suppression in HL-1 cardiomyocytes.
Purpose of the Study:
- To investigate if LF3, a known inhibitor of beta-catenin/TCF4 interaction, can counteract the suppressive effects of this signaling pathway on NaV1.5 expression in HL-1 cardiomyocytes.
- To determine LF3's impact on NaV1.5 expression, Na+ channel activity, and the beta-catenin/TCF4 interaction.
Main Methods:
- Western blotting and qRT-PCR to assess NaV1.5, beta-catenin, and TCF4 expression levels.
- Subcellular fractionation to determine NaV1.5 localization.
- Whole-cell patch-clamp recordings to measure Na+ currents and channel kinetics.
- Immunoprecipitation to confirm LF3's effect on beta-catenin/TCF4 interaction.
- Luciferase reporter assays to evaluate SCN5A promoter activity.
Main Results:
- LF3 treatment significantly increased NaV1.5 expression and Na+ currents in HL-1 cells without altering beta-catenin or TCF4 levels.
- LF3 enhanced NaV1.5 localization to the cell membrane and cytoplasm.
- Patch-clamp data showed increased Na+ currents with no significant changes in channel gating parameters.
- Immunoprecipitation confirmed that LF3 inhibits the beta-catenin/TCF4 interaction.
- Luciferase assays demonstrated LF3's ability to increase SCN5A promoter activity and reverse beta-catenin-mediated suppression.
Conclusions:
- LF3 effectively inhibits the beta-catenin/TCF4 interaction in HL-1 cardiomyocytes.
- LF3 administration leads to elevated NaV1.5 expression and enhanced cardiac Na+ channel activity.
- These findings suggest LF3 as a potential therapeutic agent for conditions involving reduced NaV1.5 expression and function.
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