Cardiac Sarcoplasmic Reticulum Calcium Cycling Proteins

描述(申请人提供)：钙循环异常，这是实验性和人类心力衰竭的普遍特征，部分原因是钙储存到肌浆网(SR)的功能受损。肌浆网钙摄取是由钙-三磷酸腺苷酶(SERCA2)介导的，其活性受磷脂蛋白(PLN)可逆调节。去磷酸化的磷蛋白抑制SERCA2，而磷酸化则解除这种抑制。在人类和实验性心力衰竭中，由于SR蛋白磷酸酶活性增加，磷蛋白高度去磷酸化，导致对SERCA2的更高抑制和心脏恶化。然而，我们最初对PLN/SERCA2复合体的简单看法已经被我们最近发现的一个由SERCA2、PLN和蛋白磷酸酶1(PP1)的两个调节因子组成的调节体所改变。PLN是蛋白质磷酸酶1(RGL)的调节亚单位，将该酶锚定在PLN上，而蛋白质磷酸酶1(PP1)的两个调节因子：抑制物-1和小热休克蛋白20(Hsp20)。HSP20在物理上与PP1相互作用并抑制其活性，导致PLN磷酸化增加 和可伸缩性。HSP20也是一种抗凋亡蛋白，可以保护心脏免受应激损伤。我们的假设是，HSP20是心脏中钙循环和细胞生存的基本调节器。HSP20水平或活性的改变将导致这些过程的中断，影响心脏重构。事实上，HSP20的一个人类突变体消除了它对钙循环和心脏保护的刺激作用。该方案的创新之处在于首次发现了HSP20/PP1/RGL/PLN/SERCA2相互作用组。我们的目标是确定破坏这种SR调节体功能的触发因素和机制，以应对衰老和临床相关的应激反应，并特别强调新发现的调节因子Hsp20。HSP20也被肾上腺素能轴磷酸化，但这种磷酸化的意义仍然难以捉摸。因此，我们的目标将首次全面描述：1)HSP20调节PP1活性、下游底物及其与抑制物-1相互作用的分子机制；2)HSP20的PKA-磷酸化在调节收缩能力和细胞存活中的功能作用，影响心肌缺血后衰老和心脏重构的过程；3)HSP20在抑制心功能障碍和病理生长方面的治疗潜力；以及4)取消HSP20对人类S10F-Hsp20突变引起的收缩和心脏保护的有益影响的途径。我们将采用分子、生物化学和生理学方法的综合方法。我们的初步研究是令人兴奋的，拟议的实验是可行的和高度相关的，因为在衰竭的心脏中蛋白磷酸酶1的水平和活性显著增加。这些发现将为调控PP1/PLN/SERCA轴、重塑和细胞凋亡的机制提供基本的见解，重点放在心力衰竭的病理生理学上，并可能揭示Hsp20作为一个新的靶点来增强应激心脏的收缩能力和细胞存活。

DESCRIPTION (provided by applicant): Abnormal Ca-cycling, which is a universal characteristic of experimental and human heart failure, is partially due to impaired calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is mediated by a Ca2+-ATPase (SERCA2), whose activity is reversibly regulated by phospholamban (PLN). Dephosphorylated phospholamban inhibits SERCA2 and phosphorylation relieves this inhibition. In human and experimental heart failure, phospholamban is highly dephosphorylated, due to increased SR protein phosphatase activity, resulting in higher inhibition of SERCA2 and cardiac deterioration. However, our initial simple view of a PLN/SERCA2 complex has been modified by our recent identification of a regulatome consisting of SERCA2, PLN, the regulatory subunit of protein phosphatase 1 (RGL), that anchors this enzyme to PLN, and the two regulators of protein phosphatase 1 (PP1): inhibitor-1 and the small heat shock protein 20 (Hsp20). Hsp20 physically interacts with PP1 and inhibits its activity, resulting in increased PLN phosphorylation and contractility. Hsp20 is also an anti-apoptotic protein protecting the heart against stress-induced injury. Our hypothesis is that Hsp20 is a fundamental regulator of calcium cycling and cell survival in the heart. Alterations in the levels or activity of Hsp20 will lead to disruption f these processes, impacting cardiac remodeling. Indeed, a human mutant of Hsp20 abrogates its stimulatory effects on Ca-cycling and cardioprotection. The innovation of this proposal is the firs identification of a Hsp20/PP1/RGL/PLN/SERCA2 interactome. Our goal is to define the triggers and mechanisms that disrupt the function of this SR regulatome in response to aging and clinically relevant stress with specific emphasis on the newly-discovered regulator, Hsp20. Hsp20 is also phosphorylated by the ¿-adrenergic axis but the significance of this phosphorylation remains elusive. Accordingly, our Aims will provide a first comprehensive characterization of the: 1) molecular mechanisms by which Hsp20 regulates PP1 activity, the downstream substrates and its interaction with inhibitor-1; 2) functional role of PKA-phosphorylation of Hsp20 in regulation of contractility and cell survival, impacting the processes of aging and cardiac remodeling, following myocardial ischemia; 3) therapeutic potential of Hsp20 in inhibition of cardiac dysfunction and pathological growth; and 4) pathways underlying abrogation of the Hsp20 beneficial effects on contractility and cardioprotection elicited by the human S10F- Hsp20 mutant. We will employ an integrative approach using molecular, biochemical and physiological methodology. Our pilot studies are exciting and the proposed experiments are feasible and highly relevant, since the levels and activity of protein phosphatase 1 are significantly increased in failing hearts. The findings will provide fundamental insights into the mechanisms regulating the PP1/PLN/SERCA axis, remodeling and apoptosis with emphasis in the pathophysiology of heart failure and may reveal Hsp20 as a new target to enhance both contractility and cell survival in the stressed heart.