Molecular Pathways of Heart K Channel Regulation

描述(申请人提供)：长期目标是揭示怀孕期间功能性肥厚和产后恢复过程中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到K+决定了啮齿动物和人类的心脏动作电位时程。在衰竭的肥厚心脏中，I-to-f分子成分，特别是Kv4.3和Kv4.2通道下调，动作电位时程增加，心律失常发生。在怀孕期间，发生心律失常的风险增加，心脏出现功能性肥大，激素水平急剧变化；然而，目前还没有关于性激素诱导的K+表达/功能变化的研究，这些研究可能解释怀孕期间的心脏风险或分娩后的可逆性。因此，主要的假设是，在怀孕和产后，性激素可能以基因组或非基因组的方式调节I对当前成分(如Kv1.4、Kv4.2、Kv4.3、KChlP2、MiRP1、频率蛋白)的调节。初步研究表明：(A)妊娠晚期心脏Kv4.3、Kv1.4和Kch/P2的转录本和Kv4.3蛋白表达减少，但Kv4.2通道转录本和Kv4.3蛋白表达减少；(B)与妊娠时一样，17-β-雌二醇(E_2)使心肌Kv4.3和Kv1.4m RNA表达减少；(C)E_2使培养的成人心肌细胞Kv4.3蛋白表达减少；(D)E_2对I-to电流具有双重作用，100 nM时(与人妊娠晚期相似)，动作电位缩短，I-to幅度增加；而10-fM-E_2则产生相反的作用；(E)c-Src酪氨酸激酶被E2激活，从肥厚开始，Kv4.3的表达减少；和(F)酪氨酸激酶的激活导致动作电位延长和电流降低。我们将主要使用小鼠和多种实验方法。本研究的具体目的是：(1)研究孕期和产后早期动作电位的重构，以及I到快(I to-f)和I到慢(I to-S)电流的重构，以及潜在的分子成分。(2)研究E_2对Ito-f/Ito-S电流及其分子组成的作用，以及E_2是否通过c-Src/MAPK(ERK)轴刺激Kv4.3基因转录。(3)对E_2非基因组调控I-to-f通道和Kv4.3通道的机制(S)进行了表征和界定。(4)研究依赖c-Src的酪氨酸磷酸化对电流和Kv4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。这些研究将为肥厚早期导致心肌K+通道重构的细胞和分子机制提供新的信息，并有助于设计新的预防医学策略。

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.