Ca2+ Waves and Arrhythmias

描述（由申请人提供）：我们计划研究的长期目标仍然和以前一样，即增加我们对心律失常机制中Ca2+波、改变的Ca和循环之间关系的理解。总体假设是，基本Ca2+释放事件（Ca2+火花）的概率增加导致事件的局部求和（宏观火花），然后通过CICR耦合来自不同sr位点的Ca2+释放，导致Ca2+波的增加，幅度，空间范围和传播速度。Ca2+波，在其幅度和程度的比例，引起延迟后去极化（DADs），因此是心律失常。我们提出，由于SR-Ca2+含量的短暂升高和心肌的不均匀性，Ca2+释放的可能性增加与几种疾病有关，并有助于心律失常。我们将使用两种方法。一种方法将是利用从心律失常性梗死心脏（IZPCs）分散的浦肯野细胞与电、荧光和共聚焦成像技术。在我们的第二种方法中，我们使用了致心律失常的多细胞小梁制剂（由于EC耦合和CHF的不均匀性）。Ca2+波的起始和传播将使用外延和共聚焦显微镜，电子显微镜检查。我们假设，在IZPCs中，不均匀的Ca2+瞬态和异常的微Ca2+波事件是由于a) Ca2+火花/事件特征与正常细胞的差异，b)亚细胞区域的SR含量差异，c)不同空间/区域类型的Ca2+释放通道（例如lP3R， RYR2, RYR3） /SERCA和NCX亚型，d)肌层下与核心区室Ca2+释放的Ca2+依赖性和对IP3R药物的敏感性差异。e)这两个细胞区室Ca2+释放对电压的响应。在Aim#2中，我们将确定Ca2+从纤维中解离是否决定了导致非均匀肌中Ca2+波和心律失常的初始Ca瞬态（浪涌）；是否Ca2+波传播需要SR Ca2+释放和扩散到邻近的SR释放位点使用外焦和共聚焦显微镜。最后，我们将确定在CHF动物的心脏小梁中Ca2+波的起始和传播阈值是否明显低于非CHF动物的肌肉。总之，这些研究将为疾病诱导的心肌细胞内Ca2+循环不均匀性、Ca2+波和心律失常之间的关系提供亚细胞基础，从而有助于确定Ca2+依赖性不可重入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.