Phospholemman and Cardiac Contracility

磷酸化和心脏收缩

• 批准号: 8255470
• 负责人: Joseph Y Cheung
• 金额: $ 38.25万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8255470
• 关键词: ATP2A2; Action Potentials; Adenoviruses; Adrenergic Agents; Adult; Affect; Alanine; Amino Acids; Angiotensin-Converting Enzyme Inhibitors; Animals; Arrhythmia; Biochemistry; Biological Models; Cardiac; Cardiac Catheterization Procedures; Cardiomyopathies; Carrier Proteins; Catecholamines; Catheterization; Cell Volumes; Cells; Co-Immunoprecipitations; Congestive Heart Failure; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Tail; Data; Disease; Dose; Echocardiography; Electrophysiology (science); Engineering; Ensure; Exhibits; Experimental Models; Family; Gene Transfer; Genes; Genetically Engineered Mouse; Grant; Hand; Health; Heart; In Vitro; Injection of therapeutic agent; Integral Membrane Protein; Ion Channel; Ion Transport; Ions; Isoproterenol; Knock-out; Knockout Mice; Laboratories; Lead; Location; Maps; Measurement; Measures; Mediating; Membrane; Mus; Muscle Cells; Mutagenesis; Myocardial Contraction; Myocardial Ischemia; Myocardium; Na(+)-K(+)-Exchanging ATPase; Performance; Pharmaceutical Preparations; Phosphoproteins; Phosphorylation; Physiological; Proteins; Radioactive Tracers; Rattus; Reagent; Regulation; Research Personnel; Resources; Rest; Risk; RyR2; Scanning; Schizophrenia; Simulate; Site; Site-Directed Mutagenesis; Skeletal Muscle; Sodium-Calcium Exchanger; Stress; Stretching; Techniques; Testing; Time; Tissues; Transgenes; Transgenic Mice; Transmembrane Domain; adrenergic; design; in vivo; member; molecular site; mutant; patch clamp; phospholamban; phospholemman; public health relevance; response; stoichiometry; transgene expression; uptake

DESCRIPTION (provided by applicant): Phospholemman (PLM), the 1st member of the FXYD family of small integral membrane proteins involved in regulation of ion transport, modulates the function of both Na+-K+-ATPase and Na+/Ca2+ exchanger (NCX1) in the heart. In the current grant period, using 3 model systems (adenovirus-mediated gene transfer in adult rat myocytes, transfected HEK293 cells, and PLM knockout (KO) mice), and different techniques (electrophysiology, biochemistry, radioactive tracer uptake, and site-directed mutagenesis), we have unequivocally established that PLM directly regulates NCX1 function, independent of its effects on Na+-K+- ATPase. We showed physical association between the cytoplasmic tails of PLM with the proximal intracellular loop of NCX1. Under conditions in which [Ca2+]o is raised to favor Ca2+ influx via NCX1, PLM regulates cardiac contractility mainly by modulating NCX1 rather than Na+-K+-ATPase activity. By contrast, when myocytes are Na+ loaded by rapid pacing and isoproterenol treatment, PLM limits inotropic response to b-adrenergic stimulation by enhancing Na+-K+-ATPase activity. Under baseline conditions, the effects of PLM on either Na+- K+-ATPase or NCX1 are not apparent. These results lead us to 2 hypotheses: (1) there is a small stretch of amino acid residues in the proximal linker domain of NCX1 that interacts with phosphorylated PLM; and (2) in the intact heart subjected to stress and therefore high catecholamine levels, PLM is phosphorylated at serine68 which simultaneously accelerates Na+-K+-ATPase but retard Na+/Ca2+ exchange activities. This coordinated action of PLM is necessary to minimize arrhythmogenesis and preserve inotropic response under b-adrenergic stimulation. In this competitive grant renewal, we wish to test our hypotheses by: (1) mapping out the exact sites/residues in NCX1 that are critically involved in its interaction with PLM; and (2) evaluate the mechanism by which PLM regulates cardiac contractility in hearts in vivo, both under resting and stressful conditions. We will use a combination of in vivo (echocardiography, catheterization, transduction of exogenous genes by rAAV9 injection) and in vitro (patch-clamp, single myocyte contractility and Ca2+ and Na+ measurements, co-immunoprecipitation and GST pulldown) techniques, focusing on 2 experimental model systems: transfected HEK293 cells and PLM-KO mice with inducible transgene (TG). We already have 2 genetically engineered mice (PLM-KO, inducible PLMS68E TG) on hand, and we are confident that given the resources, we will be able to generate inducible PLMS68E TG in PLM-KO background for our studies. We have found a NCX1 mutant (248-252 AAAAA) that exhibits normal NCX1 current but is not inhibited by PLM. By expressing this NCX1 mutant in NCX1-KO hearts (Dr. Kenneth Philipson has agreed to provide us the cardiac-specific NCX1-KO mouse), we should be able to critically test the physiological relevance of NCX1 regulation by PLM. We are the only laboratory that focuses on PLM and NCX1 interactions in the heart. We have all the reagents and techniques at hand and we believe we are well situated to critically examine the mechanism by which PLM regulates cardiac contractility. Alterations in phospholemman expression or its phosphorylation state are observed in ischemic cardiomyopathy and schizophrenia. In addition, the 2 classes of drugs (b-blockers and ACE inhibitors) that have been clinically found to be efficacious in congestive heart failure and ischemic heart disease may have PLM as a common target. Understanding how PLM regulates cardiac contractility in health and disease is therefore paramount for newer and rational therapy to be designed. PUBLIC HEALTH RELEVANCE: Phospholemman regulates 2 important ion transporters in the heart. This proposal uses 2 different genetically altered mice specifically engineered to critically evaluate whether regulation of sodium-calcium exchanger or sodium-potassium pump is the major mechanism by which phospholemman affects heart contraction. In addition, the exact molecular site in sodium-calcium exchanger interacting with phospholemman will be mapped out by mutagenesis.

描述（由申请人提供）：Phospholemman（PLM），这是与离子传输调节有关的小型整合膜蛋白的FXYD家族的第一个成员，可调节心脏中Na+-K+-ATPase和Na+-K+-ATPase和Na+-ATPase和Na+/Ca2+交换器（NCX1）的功能。在当前的赠款期内，使用3个模型系统（腺病毒介导的基因转移，转染HEK293细胞和PLM敲除（KO）小鼠）以及不同的技术（电生理学，生物化学，生物化学，放射性痕量的吸收术和现场指导的强化型），该功能不合时宜，该技术不合时宜地建立了该型独立性，该公司的功能均不在范围内。对Na+-K+ATPase的影响。我们显示了PLM的细胞质尾巴与NCX1的细胞内环之间的物理关联。在升高[Ca2+] O以通过NCX1倾向的条件下，PLM主要通过调节NCX1而不是Na+-K+-ATPase活性来调节心脏收缩。相比之下，当肌细胞通过快速起搏和异丙肾上腺素治疗加载Na+时，PLM通过增强Na+-K+-ATPase活性来限制对B-肾上腺素能刺激的肌瘤反应。在基线条件下，PLM对Na+-K+-ATPase或NCX1的影响尚不明显。这些结果使我们提出了2个假设：（1）NCX1的近端接头结构域中有一小部分氨基酸残基与磷酸化的PLM相互作用； （2）在完整的心脏受到应力和高儿茶酚胺水平的影响中，PLM在丝氨酸68处被磷酸化，同时加速Na+-K+-ATPase，但延迟Na+/Ca2+交换活动。 PLM的这种协调作用对于最大程度地减少心律失常发生并在B-肾上腺素能刺激下保留正性反应。在此竞争性授予续订中，我们希望通过以下方式测试我们的假设：（1）绘制NCX1中与PLM相互作用的确切位点/残基； （2）评估PLM调节体内心脏心脏收缩性的机制，无论是在静止和压力条件下。我们将结合体内（超声心动图，导管，通过RAAV9注射对外源基因的转导）和体外（斑块夹，单个肌细胞收缩力以及Ca2+和Na+测量值，共获得免疫原料和GST PULLDSIQUES，重点介绍2个实验模型的Hek 2 ins-trapitife and trapiely Model and transefient Models Systems： - - 跨性别模型。转基因（TG）。我们手头上已经有2只基因工程的小鼠（PLM-KO，诱导PLMS68E TG），并且我们有信心鉴于资源，我们将能够在PLM-KO背景中生成可诱导的PLMS68E TG。我们发现了一个NCX1突变体（248-252 AAAAA），该突变体表现出正常的NCX1电流，但没有被PLM抑制。通过在NCX1-KO心脏中表达该NCX1突变体（Kenneth Philipson博士已同意为我们提供心脏特异性的NCX1-KO小鼠），我们应该能够通过PLM认真测试NCX1调节的生理相关性。我们是唯一专注于心脏中PLM和NCX1相互作用的实验室。我们手头上都有所有试剂和技术，我们相信我们的位置良好，可以批判性地检查PLM调节心脏收缩性的机制。在缺血性心肌病和精神分裂症中观察到磷光体表达或其磷酸化状态的改变。此外，在临床上发现这两类药物（B阻滞剂和ACE抑制剂）在充血性心力衰竭和缺血性心脏病中具有有效性，可能具有PLM作为常见靶标。因此，了解PLM如何调节健康和疾病中的心脏收缩力对于设计新的和理性的治疗至关重要。 公共卫生相关性：磷光体调节心脏中2个重要的离子转运蛋白。该提案使用2种不同的遗传改变的小鼠，特异性地进行了精心评估的钠 - 钙交换剂的调节还是钠钾泵是磷光体人心脏收缩的主要机制。另外，将通过诱变将与磷光体相互作用的钠含量交换器中的精确分子位点绘制。