Sphingosine 1-phosphate and cardioprotection

描述（由申请人提供）：该提案基于我们之前的研究，记录了鞘氨醇激酶/鞘氨醇1-磷酸（S1P）在心肌抗损伤中的基本重要性。S1P是一种溶血磷脂，由肥大细胞、红细胞、血小板、心肌细胞和其他类型的细胞通过新生生物合成途径和质膜磷脂鞘磷脂的代谢产生。S1P的分泌导致白蛋白和血浆脂蛋白的广泛结合和纳摩尔至微摩尔浓度的循环。胞外S1P激活质膜G蛋白偶联受体S1P1、S1P2、S1P3、S1P4和S1P5。内源性和外源性S1P也获得与功能相关的细胞内浓度，并通过不明确的机制调节活力。先前的NIH拨款支持发表关于心脏组织中S1P的促生存作用的关键观察结果。正如进展报告中总结的那样，我们确定内源性S1P产生的药理激活或外源性S1P的管理足以减少缺血-再灌注小鼠心脏的梗死和增强收缩力。我们开发了一种快速准确的测量鞘氨醇激酶活性的放射测定方法，并利用药理学和基因靶向方法确定了该酶在预调节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-再氧化研究，我们发现S1P1受体功能、Akt激活以及与线粒体相互作用的细胞底物有助于S1P促进生存的作用。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、慢性病理性重塑和死亡率。本课题旨在探索S1P受体激动作用的新功能及其机制，并在动物模型上进行转化研究。在Specific Aim 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是由Robert Raffao博士提供的清道夫受体B类i型缺陷，亚形态载脂蛋白ER61 （SR- BI KO/ApoeR61h/h）小鼠。这些小鼠在高脂肪喂养下迅速发展成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Michael Mann博士提供的小鼠左冠状动脉前降支结扎引起的心室功能障碍的疗效。作为Aim 1的一部分，Julie Saba博士将合作进行转化研究，以验证抑制S1P裂解酶（催化不可逆S1P分解的酶）通过提高细胞内S1P含量导致心脏保护的假设。在Specific Aim 2中，我们将使用在Specific Aim 1中研究的模型来关注慢性S1P1受体激动作用的影响。这些研究将包括使用鞘氨醇激酶-1和-2缺失小鼠，测量下游信号通路，确定介导慢性信号的S1P受体亚型，以及评估粘附分子表达。Edward Goetzl博士将合作研究慢性激动剂暴露后心肌细胞核室中S1P1受体的定位和信号传导，Robin Shaw博士将合作研究生理和应激条件下心室肌细胞中S1P对间隙连接运输和功能的调节。公共卫生相关性：急性心脏病发作夺去许多人的生命，正如心脏病发作引起的心力衰竭一样，无论是在急性事件发生后不久还是几个月后。该建议使用多种细胞和生化技术，包括转基因小鼠，以进一步了解急性心脏损伤发生的机制，并利用这些信息，测试更有效的治疗方法。

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.