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

描述（由申请人提供）：左心室肥大（LVH）和糖尿病是心力衰竭发展的最强风险因素;此外，糖尿病与其他风险因素（如高血压）的组合显著增加心力衰竭的发生率，并降低诊断为心力衰竭的患者的生存率。启动肥大信号传导所需的关键事件是钙调磷酸酶的Ca 2+介导的激活和随后的NFAT核转位，目前认为这主要通过IP 3介导的Ca 2+从SR和核膜释放而发生。然而，在非兴奋性细胞中，广泛接受的是细胞外Ca 2+跨质膜的随后内流，所谓的“钙库操纵的钙进入”（SOCE）是激活钙调磷酸酶和NFAT易位所必需的。最近，STIM和奥赖蛋白家族已成为非兴奋细胞中SOCE的关键介质;然而，对这些蛋白在心脏中的作用知之甚少。β-N-乙酰-葡糖胺（O-GlcNAc）与丝氨酸和苏氨酸残基的O-连接连接正迅速成为许多生物过程的关键介体，并且已与糖尿病对心脏的不利影响以及SOCE的调节相关联。我们最近还表明，糖尿病损害心肌细胞肥大信号，至少部分通过增加O-GlcNAc水平。因此，基于先前关于心肌细胞中SOCE的报道，结合STIM和奥赖蛋白在介导电压非依赖性Ca 2+内流中的最新知识，结合我们对蛋白O-GlcNAc酰化的知识，我们认为，STIM 1-Orai 1促进的非电压门控Ca 2+内流是成年心肌细胞Ca 2+信号传导的关键介质，而O-STIM 1的GlcNAc酰化抑制其正常功能，从而提供高血糖症和异常Ca 2+介导的信号传导之间的联系。为了验证这一假设，我们将追求2个具体的目标：1：证明STIM 1介导的Ca 2+信号转导在成年心肌细胞中，并在体内心脏肥大的发展中发挥关键作用; 2：证明O-GlcNAc修饰的STIM 1抑制STIM 1介导的Ca 2+信号转导的正常激活，并有助于在糖尿病中观察到的肥大信号转导受损。我们将在分离的心肌细胞中使用获得和丧失功能的方法，包括一种新的诱导型心肌细胞限制性STIM 1敲除小鼠，以挑战目前公认的成年心肌细胞中Ca 2+稳态的范式。该提案的成功完成将产生重要的新见解的基本机制调节Ca 2+信号在心脏和建立第一次之间的葡萄糖代谢和Ca 2+稳态的机制联系，并确定新的分子介质的心脏肥大。

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.