Modulating Calcineurin Signaling Pathways in Muscle

描述(申请人提供)：肥厚性和缺血性心脏病在美国和全球都达到了流行的程度。因此，迫切需要新的、更有效的治疗方法。钙调神经磷酸酶是一种钙调节的蛋白磷酸酶，可促进心脏在应激状态下的肥大生长和重塑。我们的实验室发现了一个最近被重新命名为钙调神经磷酸酶调节因子的RCAN蛋白家族。我们证明了RCAN结合并抑制心肌和骨骼肌中钙调神经磷酸酶的活性。我们发现，RCAN1在转基因小鼠中的表达增加，通过抑制心肌梗死后肥大的生长和心力衰竭的进展，保护心脏免受各种压力的影响。因此，提高RCAN在心脏中的表达或活性的策略可能对心脏病的治疗和预防具有临床价值。这项建议是我们的RO1的竞争性更新，标题为“调节肌肉中的钙调神经磷酸酶信号通路”。我们最初目标的实现对理解Rcan对钙调神经磷酸酶的调节及其生物学意义有很大的贡献。我们的发现将我们带向了令人兴奋的新方向，可能将RCAN1对钙调神经磷酸酶的调节与缺氧或氧化应激反应联系起来。重要的是，我们和其他人最近发现，在缺血再灌注(I/R)后，缺乏RCAN1的小鼠的心脏梗塞面积更大。总之，这些发现表明，RCAN1既可以限制肥大生长，以应对压力超负荷，又可以减少I/R造成的损害。我们的新目标解决了从这些发现中衍生出来的重要问题。具体目的1：确定CAATT/增强子结合蛋白β(C/EBPB)在调控RCAN1.4表达中的作用，并探讨其在心脏肥厚性重构中的作用。在这一部分中，我们将测试C/EBPB的能力，它在心脏缺氧时被激活，以控制RCAN1.4的表达，并确定C/EBPB的活性是否影响钙调神经磷酸酶信号或心肌肥大。具体目标2：确定RCAN1亚型与泛素/蛋白酶体系统的相互作用，并测试这些亚型是否在心肌细胞肥大的发生中贡献了同型特异性功能。我们已经确定了两类不同的泛素连接酶复合体，它们与一种RCAN1亚型特异相互作用，但不与另一种RCAN1亚型相互作用。Cullin 4A E3连接酶复合体(Cul4A)与RCAN1.4相互作用，而作为低氧反应的组成部分的Von Hippel-Lindau因子(VHL)与RCAN1.1相互作用。具体目标3：明确RCAN1保护心肌细胞免受氧化损伤的机制。在提案的这一部分，我们将使用体外和体内技术来测试RCAN1是否保护心肌细胞免受氧化损伤，并评估AIMS 1和AIMS 2中研究的机制在这一过程中的参与。这些研究旨在阐明RCAN1的机制、调节和生理作用，并为治疗和预防心脏病的新疗法奠定基础。公共卫生相关性导致心力衰竭的心血管疾病是美国的主要死亡原因，尽管应用了最先进的治疗方法，但发病率仍在上升。显然，有必要开发新的治疗方法。这项提案将提供有关一类保护心脏的内源性蛋白家族的重要信息，该家族被称为钙调神经磷酸酶(RCAN)。这些研究将阐明rcan蛋白的机制、调节和生理作用，并为调控钙调神经磷酸酶的新治疗方法奠定基础。钙调神经磷酸酶是一种参与包括心脏病和阿尔茨海默氏症在内的广泛病理过程的蛋白质。

DESCRIPTION (provided by applicant): Hypertrophic and ischemic heart disease has reached epidemic proportions both in this country and globally. As a result, there is urgent need for new and more effective therapies. Calcineurin is a calcium-regulated protein phosphatase that promotes hypertrophic growth and remodeling of the heart under stress. Our laboratory identified a family of proteins recently renamed RCANs for Regulators of Calcineurin. We demonstrated that RCANs bind to and inhibit calcineurin activity in heart and skeletal muscle. We showed that increased expression of RCAN1 in transgenic mice protects the heart from a wide variety of stresses by blunting both hypertrophic growth and the progression to heart failure after myocardial infarction. Strategies to increase the expression or activity of RCANs in the heart may therefore have clinical value for the treatment and prevention of heart disease. This proposal is a competitive renewal of our RO1 entitled "Modulating Calcineurin Signaling Pathways in Muscle." Accomplishment of our initial goals has contributed significantly toward understanding RCAN's regulation of calcineurin and its biological significance. Our findings have taken us in exciting new directions that may link regulation of calcineurin by RCAN1 to hypoxic or oxidative stress responses. Importantly, we, and others, have recently found that the area of infarct is larger in the hearts of mice lacking RCAN1 after ischemic-reperfusion (I/R). Together these findings suggest that RCAN1 can both limit hypertrophic growth in response to pressure overload and reduce damage from I/R. Our new goals address important questions that have grown out of these findings. Specific Aim 1: To determine the role of the CAATT/enhancer binding protein beta (C/EBPb) in regulating RCAN1.4 expression and test its involvement in hypertrophic remodeling of the heart. In this section we will test the ability of C/EBPb, which is activated in response to cardiac hypoxia, to control RCAN1.4 expression and determine whether C/EBPb activity influences calcineurin signaling or cardiac hypertrophy. Specific Aim 2: To define interactions of RCAN1 isoforms with the ubiquitin/proteasome system and to test whether these contribute isoform-specific functions in the setting of cardiomyocyte hypertrophy. We have identified two different classes of ubiquitin ligase complexes that interact specifically with one RCAN1 isoform but not the other. A cullin 4A E3 ligase complex (Cul4A) interacts with RCAN1.4, whereas the Von Hippel-Lindau factor (VHL), an integral component of hypoxic responses, interacts with RCAN1.1. Specific Aim 3: To define mechanisms through which RCAN1 protects cardiomyocytes from oxidative damage. In this portion of the proposal we will use both in vitro and in vivo techniques to test whether RCAN1 protects cardiac myocytes from oxidative damage and assess the involvement of mechanisms studied in Aims 1 and 2 in this process. These studies are designed to clarify the mechanism, regulation and physiological role of RCAN1 and create the basis for novel therapeutic approaches to the treatment and prevention of heart disease. PUBLIC HEALTH RELEVANCE Cardiovascular disease leading to heart failure is the leading cause of death in the Unites States and is increasing in prevalence despite the application of state-of-the-art therapies. There is a clear need for the development of new therapies. This proposal will provide important information regarding a family of endogenous proteins that protect the heart called Regulators of Calcineurin (RCANs). These studies will clarify the mechanism, regulation and physiological role of RCAN proteins and create the basis for novel therapeutic approaches to regulating calcineurin, a protein involved in the progression of a wide range of pathologies including heart disease and Alzheimers.