Neuronal and Developmental Regulation of Pacemaker Channels

心脏交感神经支配的发生伴随着心脏离子通道功能的发育变化。特别重要的是心室起搏器电流I-f的激活电压负移的演变，在项目8和9的先前合作研究中确定。I-f之所以引起人们的兴趣，是因为（1）它在控制心率方面起着核心作用，出生时心率很快，到青春期时心率会减慢到原来的一半以下;（2）在疾病引起电流激活的正向转变的情况下，它在心率和节律控制方面起着重要作用。项目9的拟议目标来自我们早期对I-f发育变化的研究，以及在本资助期内的三项观察结果：1）I-f的发育变化部分通过培养新生儿心室肌细胞来重现。 具有交感神经或持续存在神经肽Y（NPY）和去甲肾上腺素的细胞。2)I-f在缺乏NPY的小鼠的成年心室肌细胞中比在野生型细胞中的负电压下激活，支持NPY的发育作用。3)过表达的HCN 2，一个分子相关的I-F，激活在新生儿比在成年大鼠心室肌细胞的负电压，这表明存在一个年龄特异性的因素影响门控个别HCN亚型。在这次更新申请中，项目9有四个具体目标，针对以下问题：1）发育调节I-f的NPY依赖性信号级联的组成部分是什么？2)什么额外的信号 cascade（s）有助于I-f成熟？3)I-f成熟变化的分子基础是什么？4)HCN过度表达的功能后果是什么？我们将采用大鼠和小鼠的新生和成年心室肌细胞的单细胞和细胞培养物，后者利用敲除动物来阐明相关的信号级联。将研究天然I-f和过表达的HCN电流。这些研究与交感神经支配对正常心脏发育的调节以及涉及异常神经支配和相关心律失常的疾病状态有关。此外，通过研究心肌细胞而不是异源系统中起搏通道基因的过度表达，项目9提供了一种创新的方法，该方法利用分子生物学的力量， 保留对于最终阐明这些通道如何在体内起作用以及如何在体内被控制所必需的生理背景。

The onset of cardiac sympathetic innervation is accompanied by developmental changes in cardiac ion channel function. Of particular importance is the evolution of a negative shift in activation voltage of the ventricular pacemaker current, I-f, identified in a previous collaborative study by Projects 8 and 9. I-f is of interest (1) because of its central role in the control of heart rate, which is rapid at birth and slows to less than half its original rate by adolescence, and (2) because it plays a role in rate and rhythm control in instances where disease induces a positive shift in activation of the current. The proposed aims of Project 9 derive from our earlier studies of developmental changes in I-f, as well as three observations made during the current funding period: 1) The developmental shift in I-f is partly reproduced by culturing neonatal ventricular cells with sympathetic nerves or in the sustained presence of neuropeptide Y (NPY) and norepinephrine. 2) I-f activates at less negative voltages in adult ventricular myocytes from mice lacking NPY than in cells from wild type, supporting a developmental role for NPY. 3) Over-expressed HCN2, a molecular correlate of I-f, activates at less negative voltages in neonatal than in adult rat ventricular myocytes, suggesting the existence of an age-specific factor influencing the gating of individual HCN isoforms. In this renewal application, Project 9 has four specific aims, targeted to the following questions: 1) What are the components of the NPY-dependent signaling cascade regulating I-f developmentally? 2) What additional signaling cascade(s) contributes to I-f maturation? 3) What is the molecular basis for the maturational change in I-f? 4) What are the functional consequences of HCN over-expression? We will employ single cells and cell cultures of neonatal and adult ventricular myocytes of rat and mouse, the latter to take advantage of knockout animals to elucidate the relevant signaling cascades. Both native I-f and over-expressed HCN currents will be studied. These studies are relevant to both regulation of normal cardiac developmental by sympathetic innervation and disease states involving abnormal innervation and associated arrhythmias. Further, by studying over-expression of pacemaker channel genes in myocytes rather than heterologous systems, Project 9 provides an innovative approach that uses the power of molecular biology while preserving the physiologic context essential to the ultimate elucidation of how these channels function, and can be controlled, in vivo.