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)和糖尿病是心力衰竭发展的最大危险因素；此外，糖尿病与高血压等额外危险因素的结合显著增加了心力衰竭的发生率，并降低了被诊断为心力衰竭的人的存活率。钙介导的钙调神经磷酸酶激活和随后的NFAT核转位是启动肥大信号的关键事件，目前认为这主要是通过IP3介导的从SR和核膜释放钙来实现的。然而，在不可兴奋的细胞中，人们普遍认为是细胞外钙随后跨过质膜内流，即所谓的存储操作钙进入(SOCE)，这是钙调神经磷酸酶激活和NFAT易位所必需的。最近，STIM和ORAI蛋白家族已经成为不可兴奋细胞中SOCE的关键介质；然而，人们对这些蛋白在心脏中的作用知之甚少。S-N-乙酰氨基葡萄糖(O-GlcNAc)与丝氨酸和苏氨酸残基的O-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。我们最近还表明，糖尿病损害心肌细胞肥大信号，至少部分是通过增加O-GlcNAc水平来实现的。因此，在前人对心肌细胞SOCE研究的基础上，结合STIM和OraI蛋白介导电压非依赖性钙通道的最新知识，结合我们对蛋白O-GlcN酰化的认识，我们认为STIM1-Orai1促进的非电压门控性钙通道是成年心肌细胞钙信号转导的关键介质，STIM1的O-GlcN酰化抑制其正常功能，从而在高血糖和异常的钙调节信号之间建立联系。为了验证这一假设，我们将追求两个特定的目标：1：证明STIM1介导成年心肌细胞中的钙信号并在体内心肌肥厚的发生中发挥关键作用；2：证明STIM1的O-GlcNAc修饰抑制了STIM1介导的钙信号的正常激活，并有助于糖尿病患者肥大信号的受损。我们将在分离的心肌细胞中使用功能获得和功能丧失的方法，包括一种新的可诱导的心肌细胞限制性STIM1基因敲除小鼠，以挑战目前公认的成人心肌细胞钙稳态的范例。这一提议的成功完成将对心脏中调节钙信号的基本机制产生重要的新见解，并首次建立糖代谢和钙稳态之间的机制联系，并寻找新的心肌肥厚的分子介质。

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.