Cytosolic Calcium Sweeper in Cardiac Myocytes

心肌细胞中的胞浆钙清除剂

• 批准号: 9646518
• 负责人: Hector H Valdivia
• 金额: $ 7.02万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9646518
• 关键词: Cytosolic; Calcium; Sweeper; Cardiac; Myocytes

DESCRIPTION (provided by applicant): Sorcin, a ~22-kDa Ca-binding protein widely expressed in mammalian tissues, is a novel regulator of excitation-contraction coupling in the heart. We have previously characterized the association of sorcin with the cardiac Ca release channel/ryanodine receptor (RyR2) of the sarcoplasmic reticulum. Using in vitro and in vivo functional assays, we found that sorcin 1) binds to RyR2s directly and with fast kinetics, rapidly inhibiting single channel activity, 2) undergoes Ca-dependent conformational changes, thereby modulating its affinity for yR2s, 3) localizes to z-lines in ventricular cardiomyocytes, 4) translocates from soluble to membrane bound protein targets in a Ca-dependent manner, 5) attenuates Ca sparks and Ca transients in intact cells, 6) is Phosphorylated by PKA, which in turn attenuates its inhibitory effect on RyR2s. Recently, we generated a Mouse line with genetic ablation of SRI, the gene encoding for sorcin in humans and multiple animal species. Under basal conditions, ventricular myocytes from these mice show apparently normal Ca transients and contractions. However, the apparent equilibrium in sorcin-ko mice is precarious, because a) exercise tests quickly throw these mice into aberrant cardiac electrical activity (tachyarrhythmias and sudden cardiac arrest), b) beta adrenergic stimulation of sorcin-ko hearts leads to arrhythmias and fibrillation, and c) isolated cardiomyocytes stimulated with Isoproterenol display Ca oscillations, aftercontractions, and delayed afterdepolarizations. Thus, ablation of sorcin leaves basal cardiac activity almost intact, but leads to increased automaticity. The goal of this proposal is to determine the functional role of sorcin in e-c coupling of the heart, in great part y identifying the molecular and cellular functions affected by its absence, and dissecting the mechanisms that lead to aberrant electrical behavior. We hypothesize that in normal ventricular myocytes, sorcin binds to at least four key players of e-c coupling, with its overall effect being that of a cytosolic Ca2+ sweeper. We propose: 1) to identify the molecular determinants of sorcin interaction with key proteins of e-c coupling; and 2) to determine the integrated role of sorcin in normal e-c coupling, and the pathophysiological mechanisms that lead to aberrant electrical behavior in its absence.

描述（由申请人提供）：Sorcin是在哺乳动物组织中广泛表达的〜22 kDa Ca结合蛋白，是心脏中激发反应耦合的新型调节剂。我们先前已经表征了sorcin与肌浆网的心脏CA释放通道/ryanodine受体（RYR2）的关联。 Using in vitro and in vivo functional assays, we found that sorcin 1) binds to RyR2s directly and with fast kinetics, rapidly inhibiting single channel activity, 2) undergoes Ca-dependent conformational changes, thereby modulating its affinity for yR2s, 3) localizes to z-lines in ventricular cardiomyocytes, 4) translocates from soluble to membrane bound protein targets in a CA依赖性的方式，5）减弱完整细胞中的Ca火花和Ca瞬变，6）被PKA磷酸化，进而减弱其对RYR2S的抑制作用。最近，我们生成了一条小鼠线，其遗传消融的SRI是人类和多种动物种类的sorcin的基因。在基础条件下，这些小鼠的心室肌细胞显然显示出正常的Ca瞬变和收缩。但是，sorcin-ko小鼠的明显平衡是不稳定的，因为a）运动测试迅速将这些小鼠置于异常的心脏电活动中（心律不齐 b）β肾上腺素能刺激sorcin-ko心脏会导致心律失常和纤颤，以及c）用异丙肾上腺素显示出促进，收缩和延迟的pe骨后分离的分离的心肌细胞。因此，摩西蛋白的消融几乎完好无损，但导致自动化增加。该提案的目的是确定sorcin在心脏E-C耦合中的功能作用，在很大程度上y确定了受其缺失影响的分子和细胞功能，并解剖导致异常电行为的机制。我们假设在正常的心室肌细胞中，sorcin与E-C耦合的至少四个关键参与者结合，其总体效果是细胞质Ca2+扫地剂的效果。我们提出：1）确定与E-C偶联的关键蛋白相互作用的分子决定因素； 2）确定sorcin在正常E-C耦合中的综合作用，以及在没有其不存在的情况下导致异常电行为的病理生理机制。