The Role of Phospholamban in Ischemia: Transgenic Appro*

描述(由申请人提供) 心肌顿抑描述了短暂的缺血损伤后心肌收缩能力的可逆性下降，临床表现为血运重建后泵功能恢复缓慢。实验证据表明，心肌顿抑时肌浆网(SR)功能发生改变。肌浆网钙泵(SERCA2)活性的降低和/或肌浆网钙再摄取速率的降低已经在几个物种中被描述，包括心脏手术期间遭受中度和可逆性损伤的患者。SERCA2的功能受另一种被称为磷蛋白(PLB)的SR蛋白的磷酸化状态的调节。在去磷酸化状态下，PLB抑制SERCA2活性和肌浆网钙转运。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对钙的摄取，并增加了可供释放的钙离子。父母资助的重点是确定原球蛋白的功能作用，并区分双部位原球蛋白磷酸化在生理条件下的意义，如β-肾上腺素能刺激。本次合作的主要目标是将母项目的目标延伸到心脏病。虽然有证据表明，在缺血和再灌流过程中，两个PLB残基都会被磷酸化，但每个PLB磷酸化位点在缺血再灌流心脏中的功能意义尚不清楚。这项应用提出了以下方面的研究：在顿抑心脏中，特定部位的PLB磷酸化在恢复心脏收缩和松弛能力中的作用，短暂的缺血损伤保护心脏损伤(预适应)的机制，以及这些磷酸化所涉及的信号级联反应的测定。为此，将使用在父母赠款过程中开发的具有单个位点特定突变或两个可磷酸化PLB位点同时突变的转基因模型，并结合生化测定(PLB磷酸化位点、激酶和磷酸酶活性的免疫检测)和心脏活动的机械测量。这一实验策略将使我们能够确定PLB磷酸化的意义，并区分每个PLB磷酸化位点在晕厥和预适应中的作用，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的延伸。

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.