Role of Protein Acetylation in Ncx1 Expression

描述（由申请人提供）：充血性心力衰竭是一种复杂的疾病，其中心脏不能泵出足够数量的血液来满足个体的代谢需要。它是西方世界的主要死亡原因，在美国大约有500万人（占人口的2-3%）患有这种疾病。心力衰竭是一种进行性疾病，预后很差。导致心力衰竭并推动其进展的因素是我们研究项目的重点。在分子水平上，肥厚信号介导转录激活的变化，引起基因表达的变化。这种基因表达的程序性变化对钙处理、收缩功能和代谢产生负面影响。Na+-Ca2+交换器（Ncx1）是直接影响心脏功能和生理的基因之一。Ncx1的上调直接导致SR Ca2+储存降低，收缩功能受损，去极化后延迟（DADs）的可能性更大，随后发生室性心动过速。抑制交换活性或抑制交换上调有望限制心律失常，并可能改善收缩功能。近年来的研究表明，收缩蛋白和核蛋白的乙酰化和去乙酰化在病理生理刺激下介导基因表达的快速和长期变化中起着重要的调节作用，乙酰化失衡在心血管疾病中起着重要作用。在本应用中，我们将研究组蛋白去乙酰化酶（HDAC）在Ncx1调控中的作用。我们的研究结果揭示了HDAC1/2/5在成人心脏中Ncx1转录激活所必需的独特作用，并为HDAC抑制剂的心脏保护作用提供了可能的分子解释。初步数据支持我们的假设，即HDAC1/2/5复合物使Nkx2.5脱乙酰，导致p300的募集和Ncx1启动子的上调。Aim 1中的实验将鉴定乙酰化残基，并测试乙酰化如何影响Nkx2.5与p300和Ncx1表达的相互作用。在目标2中，我们将使用ChIP和re-ChIP通过检查Ncx1转录上调过程中发生的事件的时间顺序来检验我们的假设，在目标3中，我们将研究HDAC5/9在体内的临床相关性和需求。我们的长期目标是深入了解hdac调节心脏基因表达的多种机制，并提高我们对核因子乙酰化状态如何影响转录的总体理解。这项工作将有助于我们了解hdac在心脏生理和病理生理中调控细胞事件和基因表达的作用。这些研究将有可能证明HDAC抑制剂在保持心脏肥厚的收缩功能方面的治疗潜力。公共卫生相关性：在美国，充血性心力衰竭是导致死亡的主要原因。对心脏收缩至关重要的钙在衰竭的心脏中被错误地调节。我们发现在心肌肥厚和心力衰竭的过程中，有一种调节心脏钙的蛋白是上调的。我们发现，在肥厚性心脏中，基因表达的调节因子可以阻止交换蛋白的上调。

DESCRIPTION (provided by applicant): Congestive heart failure is a complex disorder in which the heart is unable to pump a sufficient quantity of blood to meet the metabolic needs of the individual. It is the leading cause of death in the Western world with approximately 5,000,000 people (2-3% of the population) affected with this disease in the United States. Heart failure is a progressive disorder with a very poor prognosis. The factors that contribute to heart failure and drive its progress are the focus of our research program. At the molecular level, hypertrophic signals mediate change in transcriptional activation causing a change in gene expression. This programmatic change in gene expression negatively impacts calcium handling, contractile function, and metabolism. The Na+-Ca2+ exchanger (Ncx1) is one of the genes whose change in expression directly impacts cardiac function and physiology. Upregulation of Ncx1 directly results in depressed SR Ca2+ stores, impaired systolic function, and a greater potential for delayed after depolarizations (DADs), with subsequent ventricular tachycardia. Inhibition of exchanger activity or suppression of exchanger upregulation is expected to limit arrhythmias and potentially improve contractile function. In recent years, studies have shown that acetylation and deacetylation of cystolic and nuclear proteins play an important regulatory role in mediating the rapid and long-term changes in gene expression in response to pathophysiological stimuli and that acetylation imbalance can play an important role in cardiovascular disease. In this application, we will examine the role that histone deacetylases (HDAC) have in the regulation of Ncx1. Our findings reveal a unique role for HDAC1/2/5 as required for transcriptional activation of Ncx1 in the adult heart and provide a possible molecular explanation for the cardioprotective actions of HDAC inhibitors. The preliminary data support our hypothesis that the HDAC1/2/5 complex deacetylates Nkx2.5 leading to the recruitment of p300 and upregulation of the Ncx1 promoter. Experiments in Aim 1 will identify the acetylated residues and test how acetylation affects Nkx2.5 interaction with p300 and Ncx1 expression. In Aim 2 we will use ChIP and re-ChIP to test our hypothesis by examining the chronological sequence of events that occur during Ncx1 transcriptional upregulation and in Aim 3 investigate the clinical relevance and requirement of HDAC5/9 in vivo. Our long-term objectives are to provide insight into the diverse mechanisms by which HDACs regulate cardiac gene expression and improve our overall understanding regarding how the acetylation state of nuclear factors affect transcription. This work will contribute to our understanding of the role HDACs play in the regulation of cellular events and gene expression in physiology and pathophysiology of the heart. These studies will have the potential of demonstrating the therapeutic potential of HDAC inhibitors in preserving systolic function in cardiac hypertrophy. PUBLIC HEALTH RELEVANCE: Congestive heart failure is the leading cause of death in the United States. Calcium, which is critical for heart contraction is mis-regulated in the failing heart. We have found that one of the proteins regulating cardiac calcium is upregulation in cardiac hypertrophy and failure. We have found that a regulator of gene expression prevents the upregulation of the exchanger in a hypertrophic heart.