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健康和疾病中心脏自律性的机制。本提案的目的是确定在心脏发育过程中心脏起搏的离子机制。心律失常在所有年龄段都很常见，经常导致心源性猝死。大多数心律失常是心肌病的结果，有证据表明，对心律和收缩性很重要的胚胎蛋白异构体被重新表达。自发的节律性活动在心脏发育的早期就开始了，因为心脏新月折叠形成一个空心管并开始泵血。胚胎心肌细胞的相对简单性使得它在辨别胚胎和成人心脏的自律性机制方面非常有用。所有从早期胚胎心管中分离出来的肌细胞，包括原始心室，在作为单个细胞分离时都具有自律性和自动性。胚胎心脏节律性的基础尚不清楚。本实验将验证由Na/Ca交换器携带的离子电流与Ca2+、K+电流的活性协调以建立胚胎心脏起搏器舒张去极化的假设。目的是：1)确定在发育中的胚胎心脏中，心脏起搏器舒张去极化下的内向电流是否完全由INaCa携带；2)确定Ca2+电流、SR和其他来源对心脏发育中起搏器功能的贡献；3)确定lNaCa、If和K+电流在心脏发育过程中舒张期去极化产生过程中的关系。膜片钳法结合荧光钙离子和其他离子指示剂的使用，以及计算机建模，将用于确定小鼠和鸡胚胎管状心脏早期发育过程中细胞水平上的自节律机制。关键蛋白的发育表达将通过定量RT-PCR和免疫荧光共聚焦显微镜来确定。完成上述实验的结果将有助于更好地了解人类心脏病中的心律失常，并促进治疗策略的设计。

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.