Molecular Pathways of Heart K Channel Regulation

描述（由申请人提供）：长期目标是阐明妊娠期功能性肥大和产后恢复期间涉及心脏电和分子重塑的细胞和分子机制;特别是与K+通道表达/功能相关的机制。 I至K+塑造啮齿动物和人类的心脏动作电位时程。在衰竭的肥厚性心脏中，Ito-f分子组分，特别是Kv4.3和Kv4.2通道下调，增加动作电位持续时间和心肌发生。在怀孕期间，心律失常的风险增加，心脏发生功能性肥大，激素水平急剧变化;然而，没有关于性激素诱导的K+表达/功能变化的研究，可以解释怀孕期间的心脏风险或其在分娩后的可逆性。因此，主要的假设是，在怀孕期间和产后，性激素可以以基因组或非基因组方式调节I至电流组分（例如Kvl.4、Kv4.2、Kv4.3、KChlP 2、MiRP 1、frequenin）。初步研究表明：（a）心脏Kv4.3、Kv1.4和KCh/P2在妊娠晚期降低，但Kv4.2通道转录物和Kv4.3蛋白没有降低;（B）与妊娠期一样，17-β-雌二醇（E2）处理降低心脏Kv4.3和Kv1.4 mRNA;（c）E2降低培养的成人心肌细胞中Kv4.3蛋白的表达;（d）E2在100 nM时对I to电流具有双重作用（与人妊娠晚期相似），缩短动作电位，增加I ~-振幅，而10 fM E_2则相反;（e）c-Src酪氨酸激酶，其被E2激活，并且从肥大开始，减少Kv4.3的表达;和（f）酪氨酸激酶激活产生动作电位延长和I至电流减少。我们将主要使用小鼠和多种实验方法。具体目标是：（1）研究妊娠期间和产后早期动作电位、I to快（I to f）和I to慢（I to s）电流的重塑，以及潜在的分子成分。 (2)研究E2对Ito-f/Ito-s电流的作用及其分子组成，以及E2是否通过c-Src/MAPK（ERK）轴刺激Kv4.3基因转录。 (3)表征并定义E2对I to-f和Kv4.3通道的非基因组调控机制。 (4)研究c-Src依赖性酪氨酸磷酸化对I to电流和Kv 4亚型的短期和长期作用。 (5)确定Kv4.3中参与c-Src调控的基序。这些研究将为心肌肥厚早期K+通道重构的细胞和分子机制提供新的信息，并有助于预防医学的新策略的设计。

DESCRIPTION (provided by applicant): The long term goal is to unravel cellular and molecular mechanisms involved in cardiac-electrical and molecular-remodeling during functional hypertrophy in pregnancy, and recovery in postpartum; in particular, those related to K+ channel expression/function. I to K+ shapes the cardiac action potential duration in both rodents and humans. In failing hypertrophic hearts, I to-f molecular components, in particular Kv4.3 and Kv4.2 channels are downregulated increasing action potential duration and arrhythmogenesis. During pregnancy, there is an increased risk of arrhythmias, the heart develops functional hypertrophy, and hormone levels dramatically change; however, no studies are available on changes in K+ expression/function induced by sex hormones, that may explain the cardiac risks during pregnancy or their reversibility after delivery. Thus, the main hypothesis is that, during pregnancy and postpartum, sex hormones may regulate I to current components (e.g. Kvl.4, Kv4.2, Kv4.3, KChlP2, MiRP1, frequenin) in a genomic or non-genomic fashion. Preliminary Studies show that: (a) cardiac Kv4.3, Kvl.4 and KCh/P2, but not Kv4.2 channel transcripts, and Kv4.3 protein were reduced in late pregnancy; (b) as in pregnancy, 17-beta-estradiol (E2) treatment reduced cardiac Kv4.3 and Kvl.4 mRNA; (c) E2 reduced Kv4.3 protein expression in cultured adult myocytes; (d) E2 had dual effects on I to currents, at 100 nM (similar to late human pregnancy), it shortened the action potential and increased I to amplitude; whereas, an opposite effect was produced by 10 fM E2; (e) c-Src tyrosine kinase, which is activated by E2 and from the onset of hypertrophy, reduced expression of Kv4.3; and (f) tyrosine kinase activation produced action potential prolongation and I to current reduction. We will mainly use mice and multiple experimental approaches. The Specific Aims are to: (1) Investigate, during pregnancy and early postpartum, the remodeling of action potentials, and of I to fast (I to-f) and I to slow (I to-s) currents, and underlying molecular components. (2) Investigate the action of E2 on I to-f/I to-s currents and their molecular components, and if E2 stimulates Kv4.3 gene transcription through the c-Src/MAPK (ERK) axis. (3) Characterize and define the mechanism(s) of non-genomic regulation of I to-f and Kv4.3 channels by E2. (4) Investigate short- and long-term actions of c-Src-dependent tyrosine phosphorylation on I to currents and Kv4 isoforms. (5) Determine the motifs in Kv4.3 involved in its regulation by c-Src. These studies should provide new information on the cellular and molecular mechanisms leading to the remodeling of cardiac K+ channels in the early stages of hypertrophy, and help in the design of new strategies for preventive Medicine.