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潜在机制。 缺血与非缺血交界处的形成和细胞损伤 缺血心肌，即缺血的“边缘地带”。我们的假设是 气体、底物、代谢物和内皮细胞的扩散 跨越这些边界的因素导致了独特的离子和 交界区的电生理特征。我们之前的工作 而另一些人则认为，二氧化碳对phi和pho的调节可能直接 对细胞内K+损失、乳酸产生、去极化和 复极电流与肌质网钙离子释放。将这些结果综合在一起 表明二氧化碳在边界地区的扩散和积累可能 直接促成了导致发生 心律失常和细胞损伤。我们计划将重点放在胞质phi及其 并将这些变化与钙离子依赖联系起来 包括脉冲传播、自发膜的过程 去极化、传播的钙波、细胞与细胞间的电耦合 和细胞损伤。这些依赖于钙离子的效应被预测为 依赖于H+产生和跨肌膜通量。因此，二氧化碳 细胞外空间的扩散、冲刷和能量需求 预计会影响这些钙离子依赖的效应。具体目标 包括： (1)评估二氧化碳和氧气扩散以及跨肌膜H+的作用 边界带细胞内钙离子负荷的通量。(2)确定是否 缺血的心内膜下细胞层更容易发生细胞病变 去耦合、不连续脉冲传播与细胞损伤的比较 由于细胞内钙离子负荷较大，导致了壁层之间的相互作用。(三)评估 交界性室性早搏的发生机制 带，即Ca~(2+)形成和传播的pH依赖性 海浪。(4)确定血管活性多肽和内源性的作用 血管内皮细胞衍生因子对心肌细胞内pH和钙离子浓度的调节 边境区。 具体目标将通过两种缺血模型进行研究。 边界。参数特定的共聚焦荧光视频显微镜 将使用荧光探针和被动记录电极分析来 刻画CO2和O2对PHI和O2机制的影响 在单层模拟的边界区域内的Ca2+i调节 培养的新生儿心肌单核细胞。在其他实验中，共聚焦 荧光视频显微镜、硅/铱微电极和 组织学方法将使用分离的动脉灌流和 确定缺血乳头肌与升降的关系 钙离子浓度、细胞解偶联、脉冲传播和细胞损伤。