Calcium Signaling, Metabolism, and EC Coupling in Heart

申请人的长期目标是继续研究钙信号、兴奋-收缩(EC)偶联，以及这些过程对心肌能量代谢的依赖。本研究的具体目的是研究兔和小鼠(包括心脏特异性钠钙交换基因敲除小鼠)的心肌细胞：1)研究兔梗死区重塑细胞的钙信号和EC偶联。这将包括评估L钙通道功能的丧失是否可以解释EC偶联的失败，以及横管、兰尼定和二氢吡啶受体的三维结构和空间分布的显著变化是否参与了这些细胞的失败；2)研究代谢抑制对兔心室肌细胞偶联蛋白功能和结构的影响。这将包括测量一个偶联中L类钙通道的最小数量，以及代谢抑制对其功能的影响方式。特别是，作为代谢抑制期间耦联功能丧失的原因，横管系统的钙火花和尖峰形成以及细胞微结构的变化将被考虑；3)研究钠钙交换基因敲除小鼠对代谢应激的抵抗力。这将包括对代谢抑制阻止野生型小鼠反向钠钙交换激活，从而破坏钙诱导的EC偶联钙释放机制这一假说的调查。相反，假设钠钙交换基因敲除的小鼠不需要钠钙交换来进行EC偶联，因此对代谢抑制的影响具有抵抗力。我们将研究抑制钠钙交换激活对钙峰潜伏期的影响。这些实验的目的之一是解释钙离子在触发过程中的重要性。方法包括测量代谢抑制剂或受控心肌梗死治疗前后兔和小鼠的钙峰概率及其潜伏期分布。此外，这些方法还包括最近开发的重建横管系统三维结构的方法，以及在梗塞周围细胞和代谢抑制剂治疗的细胞中兰尼定和二氢吡啶受体的三维分布。与公共健康相关：研究人员正在研究使用单个心脏细胞进行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，以保存心肌功能，从而减少心力衰竭的发生率。

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.