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

描述（由申请人提供）：心脏病是美国人最大的单一杀手。越来越多的证据表明，Ca2+处理异常和心脏病的发展之间存在密切的关系。因此，了解病理条件下Ca2+信号的调控是非常重要的。Ca2+诱导的Ca2+释放（CICR）的局部控制取决于l型Ca2+通道和ryanodine受体（RyR）在双体中的空间组织。类似地，线粒体对Ca2+的摄取因其靠近Ca2+释放位点而得到促进，这是在工作变化过程中刺激氧化磷酸化所必需的过程。然而，线粒体对CICR的反馈却知之甚少。由于线粒体是活性氧（ROS）的主要来源，它们可能潜在地影响细胞质氧化还原状态，从而改变RyR打开概率。在本研究中，双光子激光显微镜系统将用于直接研究在不同实验条件下能量状态的急剧变化如何动态影响Ca2+火花特性。细胞质Ca2+（或ROS）， A^m和NADH将在分离的豚鼠心肌细胞中同时记录，并使用图像离线分析。线粒体间的时空耦合！去极化和Ca2+火花将使用定量方法进行分析。此外，将开发线粒体和Ca2+释放单元的计算模型，以定量研究线粒体能量学之间的相互作用

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.