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: 6430016
• 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: 2001-02-20 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6430016
• 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潜在机制 缺血性和非缺血性之间的边界处的形成和细胞损伤 缺血心肌，即缺血的“边界区”。我们的假设是 气体、底物、代谢物和内皮衍生物的扩散 跨越这些边界的因素负责独特的离子和 边缘区的电生理特征。我们以前的工作 其他人认为，CO2对pHi和pHo的调节可能具有直接的生物学效应， 对细胞K+损失、乳酸产生、去极化和 复极化电流和SR Ca ~（2+）释放。综合这些结果 表明边界带CO2扩散和积累可能 直接促进了导致发生 心律失常和细胞损伤。我们计划将重点放在细胞质pHi及其 调节的边界区，并将这些变化与钙依赖 包括脉冲传播、自发膜 去极化，钙波传播，细胞间电耦合 和细胞损伤。这些Ca 2+依赖性效应被预测为 依赖于H+产生和跨肌膜通量。因此，CO2 细胞外空间的扩散、冲洗和能量需求是 预测影响这些Ca 2+依赖性效应。具体目标 为： (1)评估CO2和O2扩散以及跨肌膜H+的作用 在边界区的细胞Ca 2+负荷通量。(2)以确定是否 缺血性内膜下细胞层更易受到细胞毒性的影响， 解偶联、不连续脉冲传播和细胞损伤的比较 由于细胞内Ca 2+负荷增加，导致细胞壁间层的钙离子浓度增加。(3)评价 边缘区室性早搏发生的机制 区域，即Ca 2+形成和传播的pH依赖性 波(4)以确定血管活性肽和内源性 内皮源性因子调节pHi和Ca ~（2+）i， 边境地带。 具体目标将是用两种缺血模型进行研究。 边界参数特异性共聚焦荧光视频显微镜 荧光探针和被动记录电极测定将用于 表征CO2和O2对pHi和 在单层细胞中模拟的边界区内的Ca 2 +i调节 培养的新生儿心肌单核细胞。在其他实验共焦 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， 判断缺血性乳头肌上升的关系 Ca 2 +i、细胞解偶联、脉冲传播和细胞损伤。