Wnt/beta-cantenin signaling and cardiac ion channels

 描述（由申请人提供）：离子通道活性的改变在心律失常的发生中起重要作用。由SCN 5a基因编码的NaV1.5是心脏Na+通道的亚基，其活性决定心脏兴奋性和电传导。Na+通道活性降低与不同类型心脏疾病中的心律失常有关，但该通道如何转录调节在很大程度上是未知的。FoxO 1介导氧化应激对SCN 5a启动子活性的抑制，氧化应激促进β-连环蛋白和FoxO相互作用以增强靶基因的表达。典型的Wnt/β-连环蛋白信号在正常心脏中是静止的。然而，在患病的心脏中，该信号通路被激活，而NaV1.5表达和Na+通道被抑制，这表明该信号通路可能涉及Na+通道调节。Wnt/β-catenin通路的激活降低了CK-1 β和GSK-3 β对β-catenin的磷酸化，并促进其与转录因子如TCF 4和FoxO 1的相互作用，从而调节靶基因的表达。我们的初步结果显示，用GSK-3 β抑制剂、氯化锂和BIO处理或过表达活化的β-连环蛋白导致NaV1.5表达降低。我们还发现，在HL-1心肌细胞中，组成型活性GSK-3 β S9 A的表达降低了β-连环蛋白的表达并增加了NaV1.5的表达。我们假设Wnt/β-catenin信号的激活通过与TCF 4和FoxO 1的相互作用抑制NaV 1.5的表达，导致Na+通道活性、心脏去极化和心脏传导的降低。我们将在以下3个具体目标中检验这一假设：目的1：确定β-连环蛋白在小鼠心脏中NaV1.5表达调节中的作用;目的2：确定β-连环蛋白抑制小鼠心脏中NaV1.5表达的分子机制;目的3：确定β-连环蛋白介导的心肌细胞中NaV1.5表达的抑制是否需要FoxO 1和TCF 4来调节心脏传导。为了实现这3种特异性AIM，我们将特别使用心脏特异性Cre-Lox技术，并删除小鼠心脏中的关键Wnt信号传导组分，如TCF 4、β-连环蛋白和APC以及FoxO 1转录因子。我们将鉴定这些基因工程小鼠中NaV1.5表达和Na+通道活性以及心脏电重构的改变。我们的主要预期发现是β-连环蛋白、TCF 4和FoxO 1形成复合物，通过抑制SCN 5a启动子活性来抑制NaV1.5表达，导致心脏去极化和心脏传导减慢。拟议的研究将确定一个潜在的和新的治疗靶点治疗心律失常。

 DESCRIPTION (provided by applicant): Alterations of ion channel activity play important roles in the development of cardiac arrhythmias. NaV1.5, encoded by the SCN5a gene, is a subunit of the cardiac Na+ channel, the activity of which determines cardiac excitability and electrical conduction. Decreased Na+ channel activity is linked to cardiac arrhythmias in different types of cardiac diseases, but it is largely unknown how this channel is transcriptionally regulated. FoxO1 mediates the inhibition of SCN5a promoter activity by oxidative stress, and oxidative stress promotes ß-catenin and FoxO interaction to enhance the expression of the target genes. Canonical Wnt/ß-catenin signaling is quiescent in the normal hearts. However, in the diseased hearts, this signaling pathway is activated while NaV1.5 expression and Na+ channel are inhibited, suggesting that this signaling pathway may involve the Na+ channel regulation. Activation of the Wnt/ß-catenin pathway decreases ß-catenin phosphorylation by CK- 1ß and GSK-3ß and promotes its interaction with transcriptional factors such as TCF4 and FoxO1 to regulate the target genes' expression. Our preliminary results showed that treatment with the GSK-3ß inhibitors, lithium chloride and BIO or overexpression of activated ß-catenin led to decreased NaV1.5 expression. We also found that expression of constitutively active GSK-3ßS9A decreased ß-catenin and increased NaV1.5 expression in HL-1 cardiomyocytes. We hypothesize that activation of Wnt/ß-catenin signaling suppresses NaV1.5 expression by its interaction with TCF4 and FoxO1, leading to a decrease of Na+ channel activity, cardiac depolarization and cardiac conduction. We will test this hypothesis in the 3 specific aims as follows: AIM 1: To define the role of ß-catenin in the regulation of NaV1.5 expression in mouse hearts; AIM 2: To determine the molecular mechanisms of ß-catenin suppressing NaV1.5 expression in mouse hearts; AIM 3: To determine if ß-catenin-mediated inhibition of NaV1.5 expression in cardiomyocytes requires both FoxO1 and TCF4 for cardiac conduction regulation. In order to achieve these 3 specific AIMs, we will particularly use cardiac specific Cre-Lox technology and delete key Wnt signaling components such TCF4, ß- catenin, and APC, and FoxO1 transcriptional factor in mouse hearts. We will identify the alterations of NaV1.5 expression and Na+ channel activity and cardiac electrical remodeling in these genetically engineered mice. Our main anticipated finding is that ß-catenin, TCF4 and FoxO1 form a complex to suppress NaV1.5 expression by inhibiting the SCN5a promoter activity, leading to slowed cardiac depolarization and cardiac conduction. The proposed studies will identify a potential and novel therapeutic target for treatment of cardiac arrhythmias.