MECHANISMS AND EFFECT OF CELLULAR CALCIUM ION LOADING IN THE BORDER ZONE

边缘区细胞钙离子负荷的机制和影响

• 批准号: 6564840
• 负责人: WAYNE E CASCIO
• 金额: $ 23.61万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564840
• 关键词: acidity /alkalinity; calcium flux; carbon dioxide tension; cell cell interaction; cellular respiration; confocal scanning microscopy; cytotoxicity; diffusion; electrophysiology; laboratory rabbit; laboratory rat; microelectrodes; myocardial ischemia /hypoxia; oxygen tension; sudden cardiac death; tissue /cell culture; ventricular fibrillation; video microscopy

The overall goal is the understand mechanisms underlying arrhythmia formation and cellular injury at boundaries between ischemic and non- ischemic myocardium, that is the ischemic "border zone". Our hypothesis is that diffusion of gases, substrates, metabolites and endothelial derived factors across these boundaries are responsible for the unique ionic and electrophysiologic characteristics of the border zone. Previous work by us and others suggest that modulation of pHi and pHo by CO2 may have direct effects on cellular K+ loss, lactate production, depolarizing and repolarizing current and SR Ca2+ release. Taken together these results indicate that CO2 diffusion and accumulation in the border zone might contribute directly to the mechanisms responsible for the occurrence of arrhythmias and cellular injury. We plan to focus on cytosolic pHi and its regulation in the border zone and relate these changes to Ca2+-dependent processes that include impulse propagation, spontaneous membrane depolarization, propagated calcium waves, cell-to-cell electrical coupling and cellular injury. These Ca2+-dependent effects are predicted to be dependent on H+ production and transsarcolemmal flux. As such, C02 diffusion, washout of the extracellular space and the energy demands are predicted to influence these Ca2+-dependent effects. The specific aims are: (1) to evaluate the role of CO2 and O2 diffusion, and transsarcolemmal H+ flux for cellular Ca2+ loading in the border zone. (2) to determine if the ischemic subendocardial cell layers are more susceptible to cellular uncoupling, discontinuous impulse propagation and cellular injury compared to intramural layers due to greater cellular Ca2+ loading. (3) to evaluate mechanisms for the initiation of premature ventricular beats in the border zone, namely the pH-dependence of the formation and propagation of Ca2+ waves. (4) to determine the role of vasoactive peptides and endogenous endothelial derived factors for the modulation of pHi and Ca2+i in the border zone. The specific aims will be studies with two models of ischemic boundaries. Confocal fluorescent videomicroscopy of parameter specific fluoroprobes, and passive recording electrode assays will be used to characterize the influence of CO2 and O2 on the mechanisms of pHi and Ca2+i regulation within the border zone simulated in a monolayer of cultured neonatal cardiac monocytes. In other experiments confocal fluorescent videomicroscopy, sillicon/iridium microelectrodes and histological methods will be used the isolated arterially perfused and ischemic papillary muscle to determine the relationship between the rise of Ca2+i, cellular uncoupling, impulse propagation and cellular injury.

总体目标是了解心律失常的机制 缺血性和非缺血性边界处的形成和细胞损伤 心肌缺血，即缺血的“边界区”。我们的假设是 气体、底物、代谢物和内皮衍生的扩散 跨越这些边界的因素导致了独特的离子和 边界区的电生理特征。我们之前的工作 等人认为 CO2 对 pHi 和 pHo 的调节可能具有直接作用 对细胞 K+ 损失、乳酸生成、去极化和 复极电流和 SR Ca2+ 释放。综合这些结果 表明边界地区的 CO2 扩散和积累可能 直接促成导致发生的机制 心律失常和细胞损伤。我们计划重点关注细胞质 p​​Hi 及其 边界区域的调节并将这些变化与 Ca2+ 依赖性相关 过程包括脉冲传播、自发膜 去极化、传播钙波、细胞间电耦合 和细胞损伤。这些 Ca2+ 依赖性效应预计是 取决于 H+ 的产生和跨肌膜通量。因此，C02 扩散、细胞外空间的冲刷和能量需求是 预计会影响这些 Ca2+ 依赖性效应。具体目标 是： (1)评估CO2和O2扩散以及经肌膜H+的作用 边界区细胞 Ca2+ 负荷的通量。 (2) 判断是否 缺血的心内膜下细胞层更容易受到细胞 解偶联、不连续脉冲传播和细胞损伤的比较 由于细胞内 Ca2+ 负载量较大，导致壁内层。 (3)评价 边界室性早搏的启动机制 区，即 Ca2+ 的形成和传播的 pH 依赖性 波浪。 (4)确定血管活性肽和内源性的作用 调节 pHi 和 Ca2+i 的内皮衍生因子 边境地区。 具体目标是用两种缺血模型进行研究 边界。参数特定的共焦荧光视频显微镜 荧光探针和被动记录电极测定将用于 表征 CO2 和 O2 对 pHi 机制的影响和 单层模拟边界区内的 Ca2+i 调节 培养的新生儿心脏单核细胞。在其他共焦实验中 荧光视频显微镜、硅/铱微电极和 将使用组织学方法分离动脉灌注和 判断缺血乳头肌上升的关系 Ca2+i、细胞解偶联、脉冲传播和细胞损伤。