Sphingosine 1-phosphate and cardioprotection

描述(由申请人提供)：这项建议是基于我们先前的研究，记录了鞘氨醇激酶/1-磷酸鞘氨醇(S1P)在心肌损伤抵抗中的基本重要性。S1P是一种溶血磷脂，由肥大细胞、红细胞、血小板、心肌细胞等多种细胞在新生生物合成途径和质膜磷脂鞘磷脂代谢过程中产生。S1P的分泌导致白蛋白和血浆脂蛋白的广泛结合，并在纳摩尔到微摩尔浓度下循环。细胞外S1P激活质膜G蛋白偶联受体，命名为S1P1、S1P2、S1P3、S1P4和S1P5。内源性和外源性S1P也可获得功能相关的细胞内浓度，并通过不清楚的机制调节活性。NIH之前的一项拨款支持发表关于S1P在心脏组织中的生存效应的关键观察结果。正如进展报告中总结的那样，我们确定，药物激活内源性S1P产生或给予外源性S1P足以减少缺血再灌注小鼠心脏的梗塞并增强其收缩能力。我们开发了一种快速而准确的神经鞘氨酸激酶活性的放射分析方法，并通过药理学和基因打靶的方法确定了该酶在预适应诱导的心脏保护中的重要作用。通过采用成年小鼠心室肌细胞模型进行缺氧-复氧研究，我们发现S1P1受体功能、Akt激活以及细胞底物与线粒体的相互作用有助于S1P的存活效应。已发表的研究结果和令人信服的初步结果支持了我们的中心假设，即S1P是一种有效的心脏保护介质，可以减少缺血和再灌注引起的急性组织损伤、慢性病理重塑和死亡率。目前的建议旨在探索S1P受体激动剂的新功能及其机制，并在动物模型中进行翻译研究。在具体目标1中，我们将研究选择性S1P1受体激动剂SEW 2871在两种心肌梗死模型中的作用。模型1是Robert Raffao博士提供的清道夫受体B型I类缺陷、亚型载脂蛋白ER61(SR-BI KO/ApoeR61h/h)小鼠。这些小鼠迅速发展为闭塞性冠状动脉粥样硬化、心肌梗死、心力衰竭和过早死亡。在第二个模型中，我们将研究SEW 2871对由Michael Mann博士提供的结扎左前降支所致的小鼠心功能障碍的疗效。作为目标1的一部分，Julie Saba博士将合作进行翻译研究，以测试抑制S1P裂解酶(催化不可逆转的S1P分解的酶)通过增加细胞内S1P含量而导致心脏保护的假设。在特定目标2中，我们将使用在特定目标1中研究的模型来重点研究慢性S1P1受体激动化的影响。这些研究将包括使用鞘氨醇激酶-1和-2缺失的小鼠，测量下游信号通路，确定介导慢性信号的S1P受体亚型，以及评估黏附分子的表达。Edward Goetzl博士将合作研究S1P1受体在慢性激动剂暴露后在心肌细胞核中的定位和信号传递，Robin Shaw博士将合作研究生理和应激条件下S1P对心室肌细胞缝隙连接运输和功能的调节。与公共卫生相关：急性心脏病发作夺走了许多人的生命，心脏病发作导致的心力衰竭也是如此，要么是在急性事件发生后不久，要么是在许多个月后。这项建议使用了包括转基因小鼠在内的各种细胞和生化技术，以加深我们对急性心脏损伤发生机制的理解，并利用这些信息来测试对这种疾病更有效的治疗方法。

DESCRIPTION (provided by applicant): This proposal is based on our prior studies documenting the fundamental importance of sphingosine kinase/sphingosine 1-phosphate (S1P) in myocardial resistance to injury. S1P is a lysophospholipid produced by mast cells, erythrocytes, platelets, cardiac myocytes, and other cell types in de novo biosynthetic pathways and by metabolism of the plasma membrane phospholipid sphingomyelin. Secretion of S1P leads to extensive binding by albumin and plasma lipoproteins and circulation at nanomolar to micromolar concentrations. Extracellular S1P activates plasma membrane G protein-coupled receptors designated S1P1, S1P2, S1P3, S1P4, and S1P5. Endogenous and exogenous S1P also attain functionally relevant intracellular concentrations and regulate viability through unclear mechanisms. A previous NIH grant has supported publication of key observations regarding the prosurvival effects of S1P in cardiac tissue. As summarized in the Progress Report, we determined that pharmacological activation of endogenous S1P production or administration of exogenous S1P was sufficient to decrease infarction and enhance contractility in mouse hearts subjected to ischemia-reperfusion. We developed a rapid and accurate radioassay for measurement of sphingosine kinase activity and established an essential role for the enzyme in preconditioning-induced cardioprotection using pharmacological and gene targeting approaches. By adapting an adult mouse ventricular myocyte model for hypoxia-reoxygenation studies, we showed that S1P1 receptor function, Akt activation, and cell substrates interacting with mitochondria contribute to S1P prosurvival effects. Published findings and compelling preliminary results support our central hypothesis that S1P is a potent mediator of cardioprotection that can reduce acute tissue injury, chronic pathological remodeling, and mortality caused by ischemia and reperfusion. The current proposal is designed to explore new functions of S1P receptor agonism and their mechanisms, and to perform translational studies in animal models. In Specific Aim 1, we will study effects of the selective S1P1 receptor agonist SEW 2871 in two models of myocardial infarction. Model 1 is the Scavenger Receptor Class B Type I-deficient, hypomorphic apolipoprotein ER61 (SR- BI KO/ApoeR61h/h) mouse provided by Dr. Robert Raffao. These mice rapidly develop occlusive coronary atherosclerosis, myocardial infarction, heart failure, and premature death in response to high-fat feeding. In the second model, we will study the efficacy of SEW 2871 on ventricular dysfunction caused by ligation of the left anterior descending coronary artery in mice provided by Dr. Michael Mann. As part of Aim 1, Dr. Julie Saba will collaborate on translational studies to test the hypothesis that inhibition of S1P lyase, the enzyme that catalyzes irreversible S1P breakdown, leads to cardioprotection by raising intracellular S1P content. In Specific Aim 2, we shall employ the models studied in Specific Aim 1 to focus on the effects of chronic S1P1 receptor agonism. These studies will include use of sphingosine kinase-1 and -2 null mice, measurements of downstream signaling pathways, determination of the S1P receptor subtypes that mediate chronic signals, and assessment of adhesion molecule expression. Dr. Edward Goetzl will collaborate on studies of S1P1 receptor localization and signaling in myocyte nuclear compartments after chronic agonist exposure, and Dr. Robin Shaw will collaborate in studying the regulation of gap junction trafficking and function by S1P in ventricular myocytes under physiological and stress conditions. PUBLIC HEALTH RELEVANCE: Acute heart attacks claim many lives, as does heart failure resulting from a heart attack, either soon after the acute event or many months-years later. This proposal uses a variety of cellular and biochemical techniques, including genetically altered mice, in order to further our understanding of the mechanisms by which acute heart damage occurs and using this information, to test more effective therapy for this disorder.