Calcium Signaling, Metabolism, and EC Coupling in Heart

描述（由申请人提供）：申请人的长期目标是继续研究钙信号传导、兴奋-收缩（EC）偶联以及这些过程对心肌能量代谢的依赖性。具体目的是研究来自兔和小鼠（包括心脏特异性钠-钙交换体基因敲除小鼠）的心室细胞，以：1）研究来自兔梗死周围区的重塑细胞中的钙信号传导和EC偶联。这将包括评估L型钙通道功能的丧失是否可以解释EC偶联的失败，以及这些细胞的失败是否涉及横小管、兰尼碱和二氢吡啶受体的三维结构和空间分布的显著改变; 2）研究代谢抑制对兔心室肌细胞中偶联子的功能和结构的影响。这将包括测量耦合子中L型钙通道的最小数量以及代谢抑制影响其功能的方式。特别是，将考虑作为代谢抑制期间偶联子功能丧失的原因的横小管系统的钙火花和尖峰形成和细胞微结构的改变; 3）研究钠-钙交换体敲除小鼠对代谢应激的抗性。这将包括一个假设，即代谢抑制阻止激活野生型小鼠的逆钠-钙交换，导致EC耦合的钙诱导的钙释放机制的破坏调查。相反，假设钠-钙交换体基因敲除小鼠不需要钠-钙交换来进行EC偶联，因此对代谢抑制的作用具有抗性。将检查抑制钠-钙交换激活对钙峰潜伏期的影响。这些实验，除其他外，旨在解释触发过程中的二进制裂隙钙的重要性。方法包括测量钙峰的概率和他们的潜伏期分布在兔子和小鼠治疗前后与代谢抑制剂或控制心肌梗死。此外，该方法包括最近开发的程序重建的3维结构的横小管系统和3维分布的兰尼碱和二氢吡啶受体在梗死周围细胞和细胞与代谢抑制剂处理。公共卫生相关性：研究人员正在使用单个心脏细胞研究心肌收缩的基本方面。这项工作的目的是确定在心脏病发作和心力衰竭患者中导致心肌减弱的确切机制。这将导致新的治疗策略来保护心肌功能，从而降低心力衰竭的发生率。

DESCRIPTION (provided by applicant): The applicant's long-term aims are to continue studies on calcium signaling, excitation-contraction (EC) coupling, and the dependence of these processes on energy metabolism in cardiac muscle. The specific aims are to study ventricular cells from rabbits and mice (including cardiac-specific sodium-calcium exchanger knock-out mice) to: 1) investigate calcium signaling and EC coupling in remodeled cells from the peri-infarct zone in rabbits. This will include an assessment of whether loss of L-type calcium channel function can account for failure of EC coupling and whether significant alterations in the 3-dimensional structure and spatial distribution of transverse-tubules, ryanodine and dihydropyridine receptors are involved in the failure of these cells; 2) investigate the effect of metabolic inhibition on the function and structure of couplons in rabbit ventricular myocytes. This will include a measurement of the minimum number of L-type calcium channels in a couplon and the way that metabolic inhibition affects their function. In particular, alterations in calcium spark and spike formation and cellular micro-architecture of the transverse-tubule system as a cause of the functional loss of couplons during metabolic inhibition will be considered; 3) study the resistance of sodium-calcium exchanger knock-out mice to metabolic stress. This will include an investigation of the hypothesis that metabolic inhibition prevents activation of reverse sodium-calcium exchange in wild-type mice, resulting in disruption of the calcium-induced calcium release mechanism of EC coupling. In contrast, it is hypothesized that sodium-calcium exchanger knock-out mice do not require sodium-calcium exchange for EC coupling and are therefore resistant to the effects of metabolic inhibition. The consequences of inhibiting sodium-calcium exchange activation on calcium spike latency will be examined. These experiments are, among other things, designed to explain the importance of diadic cleft calcium in the trigger process. Methods include measuring calcium spike probabilities and their latency distributions in rabbits and mice before and after treatment with metabolic inhibitors or controlled myocardial infarction. In addition the methods include recently developed procedures for reconstructing the 3-dimensional architecture of the transverse-tubule system and the 3- dimensional distribution of ryanodine and dihydropyridine receptors in peri-infarct cells and cells treated with metabolic inhibitors. PUBLIC HEALTH RELEVANCE: The investigators are studying the basic aspects of heart muscle contraction using single heart cells. The purpose of this work is to determine the exact mechanisms responsible for weakening of the heart muscle during heart attacks and in patients with heart failure. This will lead to new therapeutic strategies for preserving heart muscle function, thereby reducing the incidence of heart failure.