Mechanisms of Auto-Rhythmicity in Heart Development

描述(由申请人提供)：这项工作的长期目标是了解心脏自律性在健康和疾病中的机制。本研究的目的是确定心脏发育过程中心脏起搏的离子机制。心律失常在各个年龄段都很常见，并经常导致心脏性猝死。大多数心律失常是心肌病的结果，有证据表明，对节律性和收缩能力至关重要的胚胎蛋白亚型重新表达。自发的节律活动在心脏发育的早期就开始了，心脏新月折叠成一个中空的管子，开始泵血。胚胎心肌细胞的相对简单使其在识别胚胎和成人心脏自律性的机制方面很有用。从早期胚胎心管中分离出的所有心肌细胞，包括原始心室，都是自律性的，当分离成单个细胞时，会自发跳动。胚胎心脏节律性的基础尚不清楚。本实验将验证Na/Ca交换器携带的离子电流与Ca~(2+)、K~(2+)电流的活动相协调的假说，以建立胚胎心脏的起搏器舒张期去极化。其目的是：1)确定心脏起搏器舒张期去极化的内向电流是否仅由胚胎心脏的INaCa携带；2)确定来自钙电流、SR和其他来源的钙对心脏发育中起搏器功能的贡献；以及3)确定在心脏发育过程中产生舒张期去极化的1NaCa、IF和K电流之间的关系。膜片钳方法，结合使用荧光钙和其他离子指示剂，以及计算机模拟，将被用来确定小鼠和鸡胚胎管状心脏早期发育过程中细胞水平的自律性机制。关键蛋白的发育表达将使用定量RT-PCR和免疫荧光共聚焦显微镜来确定。拟议实验的完成结果将有助于更好地了解人类心脏病中的心律失常，并有助于治疗策略的设计。

DESCRIPTION (provided by applicant): The long term objective of this work is to understand the mechanisms for cardiac autorhythmicity in health and disease. The objective of this proposal is to determine the ionic mechanisms underlying cardiac pacemaking in developing heart. Cardiac arhythmias are common to all ages and frequently lead to sudden cardiac death. Most arhythmias are a result of cardiomyopathies in which there is evidence showing that embryonic protein isoforms, important for rhythmicity and contractility, are re-expressed. Spontaneous rhythmic activity begins very early in heart development as the cardiac crescent folds to form a hollow tube and begins pumping blood. The relative simplicity of the embryonic cardiac myocyte makes it useful in discerning mechanisms responsible for autorhythmicity in the embryonic and adult heart. All of the myocytes isolated from the early embryonic heart tube, including the primitive ventricle, are autorhythmic and beat spontaneously when isolated as single cells. The basis of rhythmicity in the embryonic heart is unknown. The proposed experiments will test the hypothesis that the ionic current carried by the Na/Ca exchanger is coordinated with the activity of Ca2+, K+ currents to establish the pacemaker diastolic depolarization in embryonic heart. The aims are: 1) determine whether the inward current underlying the cardiac pacemaker diastolic depolarization is carried exclusively by INaCa in the developing embryonic heart; 2) determine the contributions of Ca2+ from Ca2+ currents, the SR, and other sources to pacemaker function in heart development; and 3) determine the relationships between lNaCa, If and K+ currents in generating the diastolic depolarization in heart development. Patch clamp methods in conjunction with the use of fluorescent Ca and other ion indicators, and computer modeling will be used to determine the mechanisms of auto-rhythmicity at the cellular level in early development of the tubular heart in both mouse and chick embryos. Developmental expression of key proteins will be determined using quantitative RT-PCR and immunoflourescence confocal microscopy. The results from the completion of the proposed experiments will lead to a greater understanding of cardiac arhythmias in human heart disease and facilitate the design of treatment strategies.