Ca2+ Waves and Arrythmias

描述（由申请人提供）：我们计划研究的长期目标与以前一样，即增加我们对心律失常机制中Ca 2+波、改变的Ca、循环之间关系的理解。总的假设是，增加的概率的基本钙离子释放事件（钙离子火花）导致局部的事件（宏观火花）的总和，然后通过CICR耦合的钙离子释放从不同的SR网站造成钙离子波的增加，振幅，空间范围和传播速度。Ca 2+波与其振幅和范围成比例，引起延迟后除极（DADs），因此是致心律失常的。我们认为，由于SR-Ca ~（2+）含量的瞬时升高和心肌的不均匀性导致的Ca ~（2+）释放的可能性增加与多种疾病有关，并导致心律失常。我们将使用两种方法。一种方法将是使用浦肯野细胞分散从致瘤性梗死心脏（IZPC）与电气，epifluorescent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备，这是致炎性的（由于EC偶联的不均匀性和CHF）。Ca 2+波的启动和传播将使用epi和共聚焦显微镜，电子显微镜检查。目的#1我们假设IZPC中不均匀的Ca 2+瞬变和异常的微Ca 2+波事件是由于a）Ca 2+火花/事件特征与正常细胞的特征的差异，B）亚细胞区域SR含量的差异，c）不同空间/区域类型的Ca 2+释放通道（例如，IP 3R，RYR 2，RYR 3）/SERCA和NCX同种型，d）在肌膜下相对于核心区室中Ca 2+释放的Ca 2+依赖性和对IP 3R试剂的敏感性，e）在这两个细胞区室中Ca 2+释放对电压的响应。 在目标#2中，我们将确定Ca 2+从细丝解离是否决定了导致不均匀肌肉中的Ca 2+波和心律失常的初始Ca瞬变（浪涌）; Ca 2+波传播是否需要SR Ca 2+释放和扩散到相邻的SR释放部位，使用epi和共聚焦显微镜。最后，我们将确定是否启动和传播的阈值，从心脏的CHF动物的小梁中的钙波是实质上低于从动物的肌肉没有CHF。总之，这些研究将提供一个亚细胞的基础上，疾病引起的不均匀性的钙循环内的心肌细胞，钙波和心律失常之间的关系，从而将有助于确定新的分子靶点的钙依赖性非折返性心律失常。

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.