Ca2+ Waves and Arrhythmias

描述(申请人提供)：我们计划研究的长期目标一如既往，即增加我们对心律失常机制中钙波、钙变化、循环之间的关系的理解。总的假设是，基础钙释放事件(钙火花)的概率增加导致事件(宏观火花)的局部总和，然后通过CICR导致不同SR位置的钙释放的耦合，导致钙波的增加、幅度、空间范围和传播速度。Ca~(2+)波与其幅度和程度成正比，导致延迟后除极(DAD)，因此是致心律失常的。我们认为，由于SR-Ca~(2+)含量的一过性升高和心肌不均匀导致的Ca~(2+)释放增加的可能性与几种疾病有关，并与心律失常有关。我们将使用两种方法。一种方法是使用分散在致心律失常性梗死心脏(IZPC)上的浦肯野细胞，采用电学、荧光和共聚焦成像技术。在我们的第二种方法中，我们使用了一种多细胞小梁制剂，它是致心律失常的(由于EC偶联的不均匀和CHF)。钙波的启动和传播将使用表观显微镜和共聚焦显微镜、电子显微镜进行检查。目的：1我们假设IZPC细胞内不均匀的钙瞬变和异常的微钙波事件是由于a)钙火花/事件特征与正常细胞的不同，b)不同亚细胞区SR含量的差异，c)不同空间/区域类型的钙释放通道(如LP3R，RYR2，RYR3)/SERCA和NCX亚型的差异，d)这两个细胞亚室和核心室的钙释放对钙的依赖，以及对IP3R试剂的敏感性，e)这两个细胞室对钙释放的反应。在目标2中，我们将利用表观显微镜和共聚焦显微镜来确定细丝中的钙离子解离是否决定了导致不均匀肌肉中钙波和心律失常的初始钙瞬变(峰)；钙波的传播是否需要SR钙离子释放和扩散到相邻的SR释放部位。最后，我们将确定充血性心力衰竭动物心脏小梁中钙波的启动和传播阈值是否显著低于非充血性心力衰竭动物的肌肉。总之，这些研究将为疾病引起的心肌细胞内钙循环不均匀、钙波与心律失常之间的关系提供亚细胞基础，从而有助于识别钙依赖性非折返性心律失常的新分子靶点。

DESCRIPTION (provided by applicant): The long term objective of our planned studies remains as before, that is, to increase our understanding of the relationship between Ca2+ waves, altered Ca, cycling in the mechanisms of arrhythmias. The overall hypothesis is that increased probability of the fundamental Ca2+ release-event (the Ca2+ spark) leads to local summation of events(macro sparks) and, then by CICR to coupling of Ca2+ release from different SR-sites causing Ca2+ waves of increasing, amplitude, spatial extent and propagation velocity. Ca2+ waves, in proportion to their amplitude and extent, cause delayed afterdepolarizations (DADs) and therefore are arrhythmogenic. We propose that increased probability of Ca2+ release both due to transient elevations of SR-Ca2+ content and nonuniformity of the myocardium is involved in several diseases and contributes to the arrhythmias. We will use two approaches. One approach will be to use Purkinje cells dispersed from the arrhythmogenic infarcted heart (IZPCs) with electrical, epifluorescent and confocal imaging techniques. In our second approach, we use a multicellular trabecular preparation that is arrhythmogenic (due to nonuniformity of EC coupling and to CHF). The initiation and propagation of Ca2+ waves will be examined using epi- and confocal microscopy, electronmicoroscopy. Aim#1 We hypothesize that nonuniform Ca2+ transients and abnormal micro Ca2+ wave events in IZPCs are due to differences a) in Ca2+ spark/event characteristics vs. those of normal cells, b) in SR content by subcellular region, c) in different spatial/regional types of Ca2+ release channels (e g. lP3R, RYR2, RYR3) /SERCA and NCX isoforms, d) in Ca2+ dependence of Ca2+ release in the subsarcolemmal vs. core compartments and sensitivity to IP3R agents, e) in response of Ca2+ release in these two cell compartments to voltage. In Aim#2, we will determine whether Ca2+ dissociation from the filaments determines the initial Ca transient (a surge) that leads to Ca2+ waves and arrhythmias in the nonuniform muscle; whether Ca2+ wave propagation requires SR Ca2+ release and diffusion to adjacent SR release sites using epi- and confocal microscopy. Finally, we will determine whether the threshold for initiation and propagation of Ca2+ waves in trabeculae from hearts of animals with CHF is substantially lower than that in muscles from animals without CHF. In sum, these studies will provide a subcellular basis for the relationship between diseased-induced nonuniformities of Ca2+ cycling within a myocyte, Ca2+ waves and arrhythmias and thus will help in identifying new molecular targets for Ca2+ dependent nonreentrant arrhythmias.