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

描述（由申请人提供）：心脏细胞内钙信号是一个高度整合的过程，由众多通道、泵、转运体和缓冲体协同作用决定。虽然几种病理与Ca2+处理缺陷有关，但将缺陷与器官水平心功能障碍联系起来的具体机制通常没有很好的定义。其中一个例子是t波交替（TW-Alt）。TW-Alt在心电图（ECG） t波的搏动交替变化中被观察到，是一个重要的致心律失常机制，可导致心源性猝死。TW-Alt的可能性随着心动过速增加，被认为与细胞内Ca2+处理和/或细胞代谢异常有关。尽管经过多年的研究和争论，TW-Alt、心动过速、细胞内Ca2+处理和细胞代谢之间的机制联系仍不清楚。一个障碍是缺乏一个实验模型系统，在这个系统中，所有的显著因素都可以在一个综合的背景下进行探索（即工作的心脏）。为了解决这一问题，开发了一种新的方法（脉冲局部场荧光显微镜或PLFF）。这种方法允许非常局部的，高分辨率的测量表面膜电位和细胞内Ca2+处理在langendorff灌注跳动的心脏，其中器官水平参数（如心率，ECG，心室压）可以操纵和测量。本文结合多参数实时记录、心脏多个部位的同时监测和综合理论模型来确定产生TW-Alt的分子机制。本研究以小鼠心脏为研究对象，利用现有的转基因动物模型更明确地建立机制。初步结果导致了以下假设：在小鼠心脏中，心动过速（或代谢应激）期间肌浆网（SR） Ca2+摄取不足会产生细胞内Ca2+释放交替（Ca-Alt）。这种情况首先发生在心内膜细胞内，然后沿心室壁跨壁发展。这种Ca- alt产生了Na- Ca交换器活性的变化，从而产生了动作电位（AP）复极化的变化。由此产生的跨壁AP复极化差异产生了ECG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.