The Role of Phospholamban in Ischemia: Transgenic Appro*

Phospholamban 在缺血中的作用：转基因 Appro*

• 批准号: 6740936
• 负责人: Evangelia G Kranias
• 金额: $ 4.03万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6740936
• 关键词: SDS polyacrylamide gel electrophoresis; South America; adenosine triphosphate; beta adrenergic agent; biological signal transduction; calcium ion; calmodulin dependent protein kinase; disease /disorder model; enzyme activity; genetically modified animals; heart contraction; ion transport; laboratory mouse; myocardial ischemia /hypoxia; phospholamban; phosphoproteins; phosphorylation; protein kinase A; protein structure function; reperfusion; sarcoplasmic reticulum

DESCRIPTION (provided by applicant) Myocardial stunning describes the reversible decrease in myocardial contractility that follows a brief ischemic insult clinically manifested as sluggish recovery of the pump function after revascularization. Experimental evidence indicates that sarcoplasmic reticulum (SR) function is altered in the myocardial stunning. A decrease in the activity of the SR Ca2+ pump (SERCA2), and/or in the rate of Ca2+ reuptake by the SR have been described in several species, including patients submitted to moderate and reversible injury during cardiac surgery. The function of SERCA2 is regulated by the state of phosphorylation of another SR protein, named phospholamban (PLB). In the dephosphorylated state, PLB inhibits the activity of SERCA2 and the SR Ca2+ transport. Phosphorylation of PLB at Ser16 and Thr17 residues removes its inhibitory effect on SERCA2, thereby accelerating Ca2+ uptake by the SR and increasing the Ca2+ available for being released. The parent grant focuses on determining the functional role of PLB and on discriminating the significance of dual site PLB phosphorylation under physiological conditions, like beta-adrenergic stimulation. The main goal of the present collaboration constitutes an extension of the aims of the parent project to cardiac disease. Although there is evidence showing that both PLB residues become phosphorylated during ischemia and reperfusion, the functional significance of each PLB phosphorylation site in the ischemia-reperfused hearts is not known. This application proposes the study of the role of site-specific PLB phosphorylation on the recovery of cardiac contractility and relaxation in the stunned heart, and on the mechanisms by which brief ischemic insults protect from the cardiac injury (preconditioning), as well as the determination of the signaling cascade involved in these phosphorylations. To that purpose, transgenic models with specific mutation of a single site or simultaneous mutation of both phosphorylatable PLB sites, developed during the course of the parent grant, in combination with biochemical determination (immunodetection of PLB phosphorylation sites, kinase and phosphatase activities) and mechanical measurements of the cardiac activity, will be used. This experimental strategy will allow us to define the significance of PLB phosphorylation and to discriminate the role of each PLB phosphorylation site during stunning and preconditioning, which may lead to novel therapeutic approaches. This research will be done primarily in Argentina as an extension of NIH # HL26057.

描述（由申请人提供） 心肌惊人描述了心肌收缩性的可逆降低，这是在临床上短暂的缺血性侮辱性的临床表现为血运重建后泵功能的缓慢恢复。实验证据表明，肌质网（SR）功能在心肌惊人中发生了改变。 SR的活性降低了SR的Ca2+再摄取速率，包括在几种物种中，包括在心脏手术期间服用中度和可逆损伤的患者。 SERCA2的功能受另一种SR蛋白的磷酸化状态（称为Phospholamban（PLB））调节。在去磷酸化状态下，PLB抑制SERCA2和SR Ca2+转运的活性。 PLB在Ser16和Thr17残基上的磷酸化消除了其对SERCA2的抑制作用，从而加速了SR的Ca2+摄取，并增加了可用于释放的Ca2+。父母的授权专注于确定PLB的功能作用，并在生理条件下（如β-肾上腺素能刺激）区分双位点PLB磷酸化的重要性。当前合作的主要目标构成了父项目对心脏病的目标的扩展。尽管有证据表明，两个PLB残基在缺血和再灌注过程中都磷酸化，但尚不清楚缺血抑制心脏中每个PLB磷酸化位点的功能意义。该应用提出了研究部位特异性PLB磷酸化对震惊心脏收缩和放松的恢复的作用，以及对这些磷酸化涉及的信号级联的简短缺血性损伤（预处理）的简短缺血性损伤的机制。为此，在父母授予过程中开发的单个位点特异性突变或同时突变的转基因模型与生物化学测定（PLB磷酸化位点的免疫试验，激酶和磷酸酶活性的免疫试验），将使用心脏活动的机械测量。这种实验策略将使我们能够定义PLB磷酸化的重要性，并在令人惊叹和预处理过程中区分每个PLB磷酸化位点的作用，这可能导致新的治疗方法。 这项研究将主要在阿根廷进行，作为NIH＃HL26057的扩展。