Mechanisms regulating SR Ca2+ ATPase in the Atria

描述（由申请人提供）：肌浆网（SR） Ca2+ atp酶（SERCA）在Ca2+去除中起主导作用，并负责维持心脏SR Ca2+储存。SERCA泵的活性受磷蛋白（PLB）和肌磷脂（SLN）两种小分子量蛋白的调控。有充分的证据表明，PLB是心室2-肾上腺素能反应的关键介质。然而，对于SLN在心脏SR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态降低和肌细胞收缩性降低。有趣的是，在异丙肾上腺素处理和高频刺激下，SLN的抑制作用得以缓解。因此，我们的研究表明，SLN是心脏SERCA泵的一种新的调节剂。此外，我们发现SLN水平在狗和人的患病心房（心力衰竭和心律失常）中显著改变，这表明SLN /SERCA比值的改变可能导致衰竭心肌中Ca2+转运的改变。基于这些发现，我们假设SLN是心房2-肾上腺素能反应的关键介质，其表达水平的变化可能导致心房病理生理中钙稳态的改变。为了验证这些假设，我们建立了两种转基因小鼠模型1)心脏特异性SLN过表达和2)SLN敲除（KO）。目的：我将通过SLN转基因、SLN KO和PLB KO小鼠模型验证SLN是SERCA泵的主要调节剂并介导心房Ca2+运输的2-肾上腺素能调节的假设。Aim II将验证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调节的假设。SLN磷酸化的作用及其与SERCA泵的相互作用将通过定点诱变和腺病毒基因转移到心肌细胞来评估。此外，我们将确定SLN与SERCA泵的相互作用如何影响SR钙摄取动力学。Aim III将检验SLN与SERCA比值的改变是否会影响心房功能，并在负荷或压力增加时使心房易发生心房病理，包括心房颤动。我们将在SLN过表达和敲除小鼠中研究压力过载引起的心力衰竭和心脏起搏增加的影响。这些研究将为SLN在心房钙处理中的作用提供关键信息，并为确定治疗心房功能障碍（包括心房颤动）的新治疗靶点铺平道路。相关性：我们最近发现了一种新的分子，即肌磷脂。它主要存在于心脏心房，我们的研究表明，它可能调节心脏搏动过程中钙的转运。本研究计划的主要目的是了解肌磷脂如何调节钙转运和心房肌的收缩性。此外，本研究的另一个重要目标是了解其在心房病理中的作用。这些研究将采用基因工程小鼠模型来了解肌磷脂蛋白的损失是否对心功能有影响。

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.