Reorganization of calcium signaling in heart failure

描述（由申请人提供）：本研究将主要在斯洛伐克共和国进行，作为NIH资助#HL074045的延伸。本研究的总体目标是阐明哺乳动物心肌细胞双体在心力衰竭过程中钙信号通路的变化。这些结构包含l型钙通道簇（dhpr），而不是由钙离子偶联的ryanodine受体钙释放通道簇。在兴奋-收缩耦合过程中钙信号的改变导致心力衰竭的表现下降。dhpr激活钙释放过程中钙信号步骤的变化及其与双体超微结构组织和兴奋-收缩耦合单元通道拓扑结构的关系将被确定。为此，将进行全细胞钙电流、全细胞钙瞬态和二元钙释放事件（钙峰值）的动力学测量。这些测量将通过使用电子显微镜和共聚焦显微镜对管状系统、池状和纵向肌浆网的体积和表面密度以及钙通道和钙释放通道之间的共定位进行形态学分析来补充。钙电流和钙释放动力学的定量分析将用于估计钙信号的参数，这些参数对控制兴奋-收缩耦合的梯度和效率至关重要。计算机模拟实验将用于确定可能的机制，并设计具有高预测能力的实验。了解心脏E-C偶联异常在心力衰竭中是很重要的，因为它可能成为治疗干预的战略位点。此外，确定通道簇的空间组织与其钙信号传导之间的关系，对于理解突触传递、神经内分泌分泌和血管张力调节等过程具有更广泛的意义。

DESCRIPTION (provided by applicant): This research will be done primarily in Slovak Republic as an extension of NIH grant #HL074045. The overall goal of this proposal is to elucidate the changes of calcium signaling mechanisms in dyads of mammalian cardiac muscle cells during heart failure. These structures contain clusters of L-type calcium channels (DHPRs) in opposition to clusters of ryanodine receptor calcium release channels functionally coupled by calcium ions. Alterations in calcium signaling during excitation-contraction coupling contribute to the decreased performance of failing hearts. The changes in calcium signaling steps during activation of calcium release by DHPRs and their relationship to the ultrastructural organization of dyads and the topology of channels in the excitation-contraction coupling units will be determined. To this end, measurements of the kinetics of whole-cell calcium currents, whole-cell calcium transients, and dyadic calcium release events (calcium spikes) will be carried out. These measurements will be supplemented by morphological analysis of the volume and surface densities of the tubular system and of cisternal and longitudinal sarcoplasmic reticulum, and of colocalization between calcium channels and calcium release channels, using electron microscopy and confocal microscopy. Quantitative analysis of the kinetics of calcium current and calcium release in parallel will be used to estimate parameters of calcium signaling critical for control of gradation and efficiency of excitation-contraction coupling. Computer simulation of experiments will be used to ascertain possible mechanisms and to design experiments with high predictive power. Understanding the abnormalities of cardiac E-C coupling in heart failure is important since it might become a strategic site for therapeutic intervention. Furthermore, defining the relationships between the spatial organization of channel clusters and their calcium signaling has a broader significance for understanding processes such as synaptic transmission, neuroendocrine secretion and regulation of vascular tone.