Subcellular Origin of T-wave Alternans in the Beating Mouse Heart

描述(申请人提供)：心脏细胞内钙信号是一个高度集成的过程，由许多通道、泵、转运体和缓冲器的协同功能定义。虽然有几种病理机制与钙离子处理缺陷有关，但将这种缺陷与器官水平的心脏功能障碍联系起来的具体机制通常还没有很好的界定。T波交替(TW-Alt)就是一个例子。TW-Alt是指心电图中T波的交替变化，是导致心源性猝死的重要心律失常机制。TW-Alt的可能性随着心动过速的增加而增加，并被认为与细胞内钙处理和/或细胞代谢的异常有关。尽管进行了多年的研究和争论，但TW-Alt、心动过速、细胞内钙处理和细胞代谢之间的机制联系仍然不清楚。一个障碍是缺乏一个实验模型系统，其中所有显著因素都可以在一个综合的背景下(即工作心脏)进行探索。为了克服这一障碍，一种新的方法(脉冲局域荧光显微镜)被开发出来。这种方法可以非常局部化地、高分辨率地测量朗宁多夫灌流心脏跳动中的表面膜电位和细胞内钙处理，其中器官水平的参数(如心率、心电、室压)可以被操纵和测量。多参数实时记录、心脏多部位同步监测和一个完整的理论模型相结合，以确定TW-Alt产生的分子机制。在这项建议中使用了小鼠心脏，以利用现有的转基因动物模型更明确地建立机制。初步结果提出如下假设：在小鼠心脏，心动过速(或代谢应激)时肌浆网(SR)对钙的摄取不足会产生细胞内钙释放交替(Ca-Alt)。这首先发生在心内膜衬里的细胞中，然后跨室壁进展。这种Ca-Alt在Na-Ca交换活性中产生节拍变化，在动作电位(AP)中产生节拍改变。由此产生的AP复极的跨壁差异产生了在心电波形中观察到的TW-Alt。

DESCRIPTION (provided by applicant): Intracellular Ca signaling in heart is a highly integrated process defined by the concerted function of numerous channels, pumps, transporters and buffers. Although several pathologies are associated with defects in Ca2+ handling, the specific mechanisms that link the deficiency to organ-level cardiac dysfunction are generally not well defined. One example is T-wave alternans (TW-Alt). TW-Alt is observed as alternating beat- to-beat changes in the T-wave of the electrocardiogram (ECG) and constitutes an important arrhythmogenic mechanism that can lead to sudden cardiac death. Likelihood of TW-Alt increases with tachycardia and is thought to be associated with abnormalities in intracellular Ca2+ handling and/or cellular metabolism. Despite years of study and debate, the mechanistic links between TW-Alt, tachycardia, intracellular Ca2+ handling and cellular metabolism are still unclear. One obstacle has been the absence of an experimental model system where all the salient factors can be explored in an integrated context (i.e. the working heart). To address this obstacle, a novel method (pulsed local field fluorescence microscopy or PLFF) was developed. This method allows very localized, high-resolution measurements of surface membrane potential and intracellular Ca2+ handling in Langendorff-perfused beating hearts where organ-level parameters (like heart rate, ECG, ventricular pressure) can be manipulated and measured. Multiple parameter real-time recording, simultaneous monitoring at multiple sites on the heart, and an integrative theoretical model are combined here to identify the molecular mechanisms that generate TW-Alt. Mouse heart is used in this proposal to more definitively establish mechanism using existing transgenic animal models. Preliminary results have led to the following hypothesis: In the mouse heart, insufficient Ca2+ uptake by the sarcoplasmic reticulum (SR) during tachycardia (or metabolic stress) produces intracellular Ca2+ release alternans (Ca-Alt). This first occurs in cells lining the endocardium and then progresses transmurally across the ventricular wall. This Ca-Alt generates beat-to-beat changes in Na- Ca exchanger activity producing beat-to-beat alterations in action potential (AP) repolarization. The resulting transmural difference in AP repolarization creates the TW-Alt observed in the ECG waveform.