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缺血性损伤后心肌收缩力的可逆性降低，临床表现为血运重建后泵功能恢复缓慢。实验证据表明，肌浆网（SR）功能的改变，在心肌顿抑。SR钙泵（SERCA 2）活性降低和/或SR钙再摄取速率降低已在几个种属中描述，包括心脏手术期间遭受中度和可逆性损伤的患者。SERCA 2的功能受另一种SR蛋白磷酸化状态的调节，称为受磷蛋白（PLB）。在去磷酸化状态下，PLB抑制SERCA 2的活性和SR Ca 2+转运。PLB在Ser 16和Thr 17残基处的磷酸化消除了其对SERCA 2的抑制作用，从而加速SR对Ca 2+的摄取并增加可用于释放的Ca 2+。父母补助金的重点是确定PLB的功能作用，并在生理条件下，如β-肾上腺素能刺激双位点PLB磷酸化的意义进行区分。目前合作的主要目标是将母项目的目标扩展到心脏病。虽然有证据表明，两个PLB残基在缺血和再灌注期间变得磷酸化，但每个PLB磷酸化位点在缺血-再灌注心脏中的功能意义尚不清楚。本申请提出了研究位点特异性PLB磷酸化对休克心脏中心脏收缩力和舒张的恢复的作用，以及短暂缺血性损伤保护心脏损伤（预处理）的机制，以及确定参与这些磷酸化的信号级联。为此，将使用在母基金资助期间开发的具有单个位点特异性突变或两个可磷酸化PLB位点同时突变的转基因模型，并结合生物化学测定（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。这种实验策略将使我们能够定义PLB磷酸化的意义，并区分每个PLB磷酸化位点在击昏和预处理过程中的作用，这可能会导致新的治疗方法。 这项研究将主要在阿根廷进行，作为NIH #HL 26057的扩展。

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.