Mechanisms regulating SR Ca2+ ATPase in the Atria

描述(申请人提供)：肌浆网(SR)钙ATPase(SERCA)在清除钙中起主导作用，负责维持心脏肌浆网(SR)的钙储存。SERCA泵的活性受磷蛋白(PLB)和肌磷脂(SLN)两种小分子蛋白的调节。已有文献表明，PLB是脑室内2-肾上腺素能反应的关键介质。然而，SLN在心脏肌浆网钙稳态中的作用知之甚少。我们实验室最近的研究表明，SLN主要在心房表达。SLN在心室肌细胞中的异位表达导致钙瞬变和心肌细胞收缩能力降低。有趣的是，在高频刺激和异丙肾上腺素处理时，SLN的抑制作用被解除。因此，我们的研究表明SLN是一种新的心脏SERCA泵调节因子。此外，我们还发现，在犬和人的病变心房(心力衰竭和心律失常)中，SLN水平显著改变，这表明SLN/SERCA比率的改变可能有助于衰竭心肌钙转运的改变。基于这些发现，我们假设SLN是心房中2-肾上腺素能反应的关键介质，其表达水平的变化可能有助于心房病理生理学中所见的钙稳态的改变。为了验证这些假设，我们建立了两个转基因小鼠模型：1)心脏特异性SLN的过度表达；2)SLN基因敲除(KO)。目的利用SLN转基因、SLN KO和PLB KO小鼠模型，验证SLN是SERCA泵的主要调节因子，并介导2-肾上腺素能调节心房钙转运的假说。目的验证SLN对SERCA泵的作用是直接的，其抑制功能受磷酸化和去磷酸化状态的调节。SLN的磷酸化作用及其与SERCA泵的相互作用将通过定点突变和腺病毒基因转移到心肌细胞来评估。此外，我们还将确定SLN与SERCA泵的相互作用如何影响SR钙摄取的动力学。目的III验证SLN/SERCA比值改变会影响心房功能的假说，并在负荷或应激增加时使心房易发生包括心房颤动在内的心房病变。我们将在SLN过度表达和基因敲除小鼠中研究压力超负荷导致的心力衰竭和心脏起搏增加的影响。这些研究将提供关于SLN在心房钙调节中的作用的重要信息，并为确定治疗包括心房颤动在内的心房功能障碍的新的治疗靶点铺平道路。相关性：我们最近发现了一种新的分子，即肌磷脂。它主要存在于心脏的房室，我们的研究表明，它可能在心脏搏动期间调节心脏的钙转运。这项研究计划的一个主要目标是了解Sarcolipin如何调节心房肌的钙转运和收缩能力。此外，本研究的另一个重要目的是了解其在心房病理中的作用。这些研究将使用基因工程小鼠模型来了解Sarcolipin蛋白的丢失是否会对心脏功能产生影响。

DESCRIPTION (provided by applicant): The Sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) plays a dominant role in Ca2+ removal and is responsible for maintaining cardiac SR Ca2+ store. The activity of SERCA pump is regulated by two small molecular weight proteins, phospholamban (PLB) and sarcolipin (SLN). It is well documented that PLB is the key mediator of 2-adrenergic response in the ventricle. However, little is known about the role of SLN in cardiac SR calcium homeostasis. Recent studies from our laboratory have shown that SLN is predominantly expressed in the atria. Ectopic expression of SLN in the ventricular myocytes resulted in decreased calcium transients and myocyte contractility. Interestingly, the inhibitory effect of SLN was relieved upon isoproterenol treatment and stimulation at high frequency. Our studies therefore suggest that SLN is a novel regulator of cardiac SERCA pump. In addition, we found that SLN levels are significantly altered in diseased atria (heart failure and arrhythmia) of dogs and human, suggesting that an alteration in SLN /SERCA ratio could contribute to altered Ca2+ transport in failing myocardium. Based on these findings, we hypothesize that SLN is a key mediator of 2-adrenergic response in the atria and changes in its expression level may contribute to altered calcium homeostasis seen in atrial pathophysiology. In order to test these hypotheses, we have generated two transgenic mouse models 1) cardiac-specific over expression of SLN and, 2) SLN knockout (KO). Aim I will test the hypothesis that SLN is the major regulator of SERCA pump and mediates the 2-adrenergic regulation of Ca2+ transport in atria using SLN transgenic, SLN KO and PLB KO mouse models. Aim II will test the hypothesis that SLN action on SERCA pump is direct and its inhibitory function is regulated by phosphorylation and dephosphorylation status. The role of SLN phosphorylation and its interaction with SERCA pump will be assessed using site directed mutagenesis, and adenoviral gene transfer into cardiac myocytes. In addition, we will determine how SLN interaction with SERCA pump affect the kinetics of the SR calcium uptake. Aim III will test the hypothesis that alterations in SLN to SERCA ratio will affect atrial function and predispose the atria to develop atrial pathology including atrial fibrillation upon increased load or stress. We will study the effect of pressure overload induced heart failure and increased pacing of the heart in SLN overexpressing and knockout mice. These studies will provide critical information on the role of SLN in atrial calcium handling and pave the way towards identifying novel therapeutic targets for treating atrial dysfunction including atrial fibrillation. RELEVANCE: We recently identified a novel molecule namely sarcolipin. It is found predominantly in the atrial chamber of the heart and our studies indicate that it may regulate cardiac calcium transport during the beat to beat function of the heart. A major goal of this research proposal is to understand how Sarcolipin regulates Calcium transport and contractility of the atrial muscle. In addition another important goal of this study is to understand its role in atrial pathology. These studies will employ genetically engineered mouse models to understand if loss of Sarcolipin protein has an effect on cardiac function.