Investigate the effect of mitochondrial energy state on Ca2+ sparks and handling

描述(申请人提供)：心脏病是美国人的最大杀手。越来越多的证据表明，钙调节异常与心脏病的发生密切相关。因此，了解病理条件下钙信号的调控是非常重要的。钙诱导钙释放的局部调控依赖于二联体中L类钙通道和兰尼定受体的空间构筑。类似地，线粒体的钙摄取是因为它们非常接近钙释放位点，这是在做功变化期间刺激氧化磷酸化所必需的过程。然而，线粒体对CICR的反馈还不太清楚。由于线粒体是活性氧物种(ROS)的主要来源，它们可能潜在地影响细胞质的氧化还原状态，进而改变RyR开放的概率。在这项拟议的研究中，将使用一个双光子激光显微镜系统来直接研究在不同的实验条件下，能量状态的急剧变化如何动态地影响钙离子的火花特性。在分离的豚鼠心肌细胞上，同时记录细胞内钙离子(或ROS)、A^m和NADH，并进行离线成像分析。线粒体之间的时空耦合！去极化和钙火花将用定量的方法进行分析。此外，还将建立线粒体和钙释放单位的计算模型，以定量研究线粒体能量学之间的相互作用 和当地的钙离子处理。最后，建立了心肌细胞与底物结合的完整模型 新陈代谢、细胞电生理学、pH调节和E-C偶联将被用来研究缺血-再灌注期间能量产生、离子通道、钙处理和pH变化以及由此导致的心脏收缩功能降低的机制。通过将实验和计算结果相结合，这些研究将使我们能够全面了解缺血后损伤的起源和心力衰竭的发展，并极大地促进心脏疾病新疗法的发展。

DESCRIPTION(provided by applicant): Heart disease is the single largest killer of the American. A growing body of evidence has shown that there is a close relationship between Ca2+ handling abnormalities and development of heart disease. Therefore it is fundamentally important to understand the regulation of Ca2+ signaling under pathological conditions. Local control of Ca2+-induced Ca2+ release (CICR) depends on the spatial organization of L-type Ca2+ channels and ryanodine receptors (RyR) in the dyad. Analogously, Ca2+ uptake by mitochondria is facilitated by their close proximity to the Ca2+ release sites, a process required for stimulating oxidative phosphorylation during changes in work. Mitochondrial feedback on CICR, however, is less well understood. Since mitochondria are a primary source of reactive oxygen species (ROS), they could potentially influence the cytosolic redox state, in turn altering RyR open probability. In this proposed study, a two photon laser microscope system will be used to directly examine how acute changes in energy state dynamically influence Ca2+ spark properties under various experimental conditions. Cytosolic Ca2+ (or ROS), A^m, and NADH will be recorded simultaneously in isolated guinea pig cardiomyocytes and analyzed offline using imaged. The spatiotemporal coupling between mitochondria! depolarization and Ca2+ sparks will be analyzed using a quantitative approach. Furthermore, a computational model of mitochondria and Ca2+ release unit will be developed to quantitatively investigate the interaction between mitochondrial energetics and local Ca2+ handling. Finally, an integrated model of the cardiomyocyte incorporating substrate metabolism, cellular electrophysiology, pH regulation and E-C coupling will be developed to investigate the mechanisms underlying alterations in energy production, ion channels, Ca2+ handling and pH, as well as the resulting reduction of cardiac contractile function during ischemia-reperfusion. By combining the experimental and computational results, these studies will allow for a complete understanding the origin of post-ischemic injury and development of heart failure, and significantly spur the development of novel heart disease therapies.