MECHANISMS AND CONSEQUENCES OF INTRACELLULAR CALCIUM OVERLOAD IN HEART

细胞内Ca（Cai）超载是一种众所周知的促凋亡因子 其通过促进自律性而导致局灶性心律失常， 触发活动，并通过促进细胞解偶联来重返大气层 和缓慢的传导。 作为心肌缺血的后遗症， 再灌注时，Cai过载导致细胞损伤和心律失常， 包括心室纤颤 然而，细胞机制 在缺血/再灌注期间负责Cai超负荷的仍然存在 不完全理解。 这项建议的目的是 表征代谢抑制的亚细胞机制， 缺血环境的成分改变了Cai的调节， 心室肌细胞 Cai（使用fura-2），膜电流和电压， 在离体兔中同时监测细胞缩短 和豚鼠心室肌细胞全细胞膜片钳技术 暴露于代谢抑制剂（联合或选择性）期间的状况 糖酵解和氧化代谢的抑制）以及各种 缺血环境的组分（例如氧自由基和 两亲物）。 使用快速细胞外溶液交换装置， 促进药物干预和离子取代，我们将 详细描述这些干预措施对个人的影响， 兴奋-收缩耦合的组成部分，负责调节 CaI包括钙电流（L型和T型）、Na-Ca交换、 肌浆网、线粒体和肌膜钙泵。 我们 还将在巨大的切除膜贴片上进行研究，以评估 缺血环境成分对钠钙交换的影响。 一 第二个主要目标是探索蔡超载的机制 会导致心律失常 特别是，我们将测试一个假设预测 从所描述的心脏动作电位的计算机模拟， 在项目3中;即，通过延迟或加速舒张 去极化，延迟后去极化由于蔡超载是 能够在单个肌细胞中产生混乱的心律失常。 如果 在分离的心肌细胞中可以产生混乱的心律失常， 适用于“混沌控制”，使用 项目3，这将使我们能够研究混沌控制的机制 在细胞和亚细胞水平上使用膜片钳和荧光 指标技术 这些研究将提供重要的新 有关Cai调节异常发病机制信息， 心肌缺血/再灌注及其致炎性后果。

Intracellular Ca (Cai) overload is a well-known arrhythmogenic factor which contributes to focal arrhythmias by promoting automaticity and triggered activity, and to reentry by facilitating cellular uncoupling and slow conduction. As a sequela of myocardial ischemia and reperfusion, Cai overload contributes to cellular injury and arrhythmias, including ventricular fibrillation. However, the cellular mechanisms responsible for Cai overload during ischemia/reperfusion remain incompletely understood. The objective of this proposal is to characterize the subcellular mechanisms by which metabolic inhibition and components of the ischemia environment alter Cai regulation in isolated ventricular myocytes. Cai (using fura-2), membrane current and voltage, and cell shortening will be monitored simultaneously in isolated rabbit and guinea pig ventricular myocytes under whole cell patch clamp conditions during exposure to metabolic inhibitors (combined or selective inhibition of glycolysis and oxidative metabolism) and to various components of the ischemic environment (e.g. oxygen free radicals and amphiphiles). Using a rapid extracellular solution exchange device to facilitate pharmacologic interventions and ionic substitutions, we will characterize in detail the effects of these interventions on individual components of excitation-contraction coupling responsible for regulating Cai, including the Ca current (L and T types), Na-Ca exchange, the sarcoplasmic reticulum, mitochondria and the sarcolemmal Ca pump. We will also perform studies in giant excised membrane patches to assess the effects of components of the ischemic environment on Na-Ca exchange. A second major goal is to explore the mechanisms by which Cai overload causes arrhythmias. In particular, we will test a hypothesis predicted from the computer simulations of the cardiac action potential described in Project 3; namely that, by delaying or accelerating diastolic depolarization, delayed afterdepolarizations due to Cai overload are capable of producing a chaotic arrhythmia in a single myocyte. If a chaotic arrhythmia can be produced in the isolated myocyte, it may be amenable to 'chaos control' using the pacing algorithm described in Project 3, which will allow us to study the mechanisms of chaos control at a cellular and subcellular level using patch-clamp and fluorescent indicator techniques. These studies will provide important new information about pathogenesis of abnormal Cai regulation relevant to myocardial ischemia/reperfusion, and its arrhythmogenic consequences.