STIM1 mediated calcium entry: A new paradigm of metabolic regulation of cardiomyo

描述（由申请人提供）：左心室肥厚（LVH）和糖尿病是发展为心力衰竭的最有效的危险因素；此外，糖尿病与其他危险因素（如高血压）的合并显著增加心力衰竭的发生率，并降低心力衰竭患者的生存率。肥厚性信号起始所需的一个关键事件是Ca2+介导的钙调磷酸酶激活和随后的NFAT核易位，目前认为这主要是通过IP3介导的SR和核膜Ca2+释放发生的。然而，在不可兴奋的细胞中，人们普遍认为是细胞外Ca2+随后穿过质膜流入，即所谓的“储存操作钙进入”（SOCE），这对于钙调磷酸酶和NFAT易位的激活是必不可少的。最近，STIM和Orai蛋白家族已成为不可兴奋细胞中SOCE的关键介质；然而，人们对这些蛋白质在心脏中的作用知之甚少。ss- n -乙酰氨基葡萄糖（O-GlcNAc）与丝氨酸和苏氨酸残基的o键连接正迅速成为许多生物过程的关键介质，并与糖尿病对心脏的不利影响以及SOCE的调节有关。我们最近也表明，糖尿病损害心肌细胞肥厚信号，至少部分是由于O-GlcNAc水平升高。因此，基于先前关于心肌细胞中SOCE的报道，结合STIM和Orai蛋白介导电压无关Ca2+进入的最新知识，结合我们对o - glcnac酰化蛋白的了解，我们提出STIM1- orai1促进非电压门控Ca2+进入是成人心肌细胞Ca2+信号传导的关键介质，并且STIM1的o - glcn酰化抑制其正常功能，从而在高血糖和异常Ca2+介导的信号传导之间提供联系。为了验证这一假设，我们将追求两个特定的目标：1：证明STIM1介导成人心肌细胞中的Ca2+信号传导，并在体内心脏肥大的发展中发挥关键作用；2：证明O-GlcNAc修饰STIM1抑制STIM1介导的Ca2+信号的正常激活，并有助于糖尿病中肥大信号的受损。我们将在分离的心肌细胞中使用功能的获得和丧失方法，包括一种新型的可诱导心肌细胞限制性STIM1敲除小鼠，以挑战目前公认的成人心肌细胞中Ca2+稳态的范式。这一建议的成功完成将对心脏中Ca2+信号调节的基本机制产生重要的新见解，并首次建立葡萄糖代谢和Ca2+稳态之间的机制联系，并确定心脏肥厚的新分子介质。

DESCRIPTION (provided by applicant): Left ventricular hypertrophy (LVH) and diabetes are among the most potent risk factors for the development of heart failure; furthermore, the combination of diabetes with additional risk factors such as hypertension markedly increases the incidence of heart failure and decreases survival of those diagnosed with heart failure. A key event required for the initiation of hypertrophic signaling is the Ca2+ mediated activation of calcineurin and subsequent nuclear translocation of NFAT, which is currently believed to occur primarily via IP3 mediated Ca2+ release from the SR and nuclear envelope. However, in non-excitable cells, it is widely accepted that it is the subsequent influx of extracellular Ca2+ acros the plasma membrane, so called "store operated calcium entry" (SOCE) that is essential for activation of calcineurin and NFAT translocation. Recently STIM and Orai protein families have emerged as critical mediators of SOCE in non-excitable cells; however, little is known about the role of these proteins in the heart. The O-linked attachment of ss-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is rapidly emerging as a key mediator of numerous biological processes and has been linked to the adverse effects of diabetes on the heart and also to the regulation of SOCE. We have also recently shown that diabetes impairs cardiomyocyte hypertrophic signaling, at least in part by increased O-GlcNAc levels. Therefore, building on previous reports of SOCE in cardiomyocytes, integrating the recent knowledge of STIM and Orai proteins in mediating voltage- independent Ca2+ entry, combined with our knowledge of protein O-GlcNAcylation, we propose that STIM1-Orai1 facilitated non-voltage gated Ca2+ entry is a key mediator of Ca2+ signaling in adult cardiomyocytes and that O-GlcNAcylation of STIM1 inhibits its normal function thus providing a link between hyperglycemia and abnormal Ca2+-mediated signaling. To test this hypothesis we will pursue 2 specific aims: 1: Demonstrate that STIM1 mediates Ca2+ signaling in adult cardiomyocytes and plays a key role in development of cardiac hypertrophy in vivo; 2: Demonstrate that O-GlcNAc modification of STIM1 inhibits normal activation of STIM1-mediated Ca2+ signaling and contributes to impaired hypertrophic signaling seen in diabetes. We will use gain and loss of function approaches in isolated cardiomyocytes including a novel inducible cardiomyocyte restricted STIM1 knockout mouse to challenge the currently accepted paradigm of Ca2+ homeostasis in adult cardiomyocytes. The successful completion of this proposal will yield significant new insights into the fundamental mechanisms regulating Ca2+ signaling in the heart and establish for the first time a mechanistic link between glucose metabolism and Ca2+ homeostasis and identify novel molecular mediators of cardiac hypertrophy.