Ryanodine receptor in cardiac hypertrophy and heart failure

瑞尼定受体在心脏肥大和心力衰竭中的作用

• 批准号: 7582580
• 负责人: GERHARD W MEISSNER
• 金额: $ 36.95万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7582580
• 关键词: Action Potentials; Aged, 80 and over; Binding; Biological Assay; Birth; Body Weight; Calcineurin; Calmodulin; Cardiac; Cardiac Myocytes; Cause of Death; Cells; Docking; Embryo; Embryonic Heart; Event; Gene Proteins; Genes; Goals; Growth; Heart; Heart Hypertrophy; Heart Rate; Heart failure; Histone Deacetylase; Human; Immunoblot Analysis; Immunoblotting; Immunoprecipitation; Intracellular Membranes; Ion Channel; Ions; Kidney; Luciferases; Measurement; Measures; Membrane; Messenger RNA; Morphology; Mus; Mutant Strains Mice; Mutation; Myocardium; Newborn Infant; Patch-Clamp Techniques; Phosphorylation; Phosphorylation Site; Preparation; Protein Kinase C; Proteins; Regulation; Relative (related person); Research; Reverse Transcriptase Polymerase Chain Reaction; Risk; RyR2; Ryanodine; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Tacrolimus Binding Proteins; Testing; Transgenes; Up-Regulation; Vesicle; base; calmodulin-dependent protein kinase II; cellular imaging; extracellular; mutant; myocyte-specific enhancer-binding factor 2; novel strategies; promoter; public health relevance; response; transcription factor; uptake

DESCRIPTION (provided by applicant): The goal of our proposed research is to understand how the cardiac Ca2+ release channel (ryanodine receptor, RyR2) regulates cardiac function. The RyR2s are 2,200 kDa ion channels that release Ca2+ ions in response to an action potential from the sarcoplasmic reticulum. The RyR2 ion channel is composed of four RyR2 560 kDa subunits that bind calmodulin (CaM), and four small 12.6 kDa FK506 binding proteins. Our proposed studies make use of genetically modified mice with mutations in the CaM binding domain of RyR2 to elucidate signaling mechanisms associated with cardiac hypertrophy. Homozygous mice expressing a mutant form of RyR2 (RyR2-W3587A/L3591D/F3603A or RyR2ADA), that is not inhibited by CaM at diastolic and systolic Ca2+ concentrations, show signs of cardiac hypertrophy as early as 1 day after birth. There is up- regulation of genes and proteins associated with class II histone deacetylase(HDAC)/myocyte enhancer factor- 2(MEF2) and calcineurin signaling pathways, and the homozygous mutant mice die within two weeks of birth. Genetically modified mice deficient in CaM regulation of RyR2 by CaM at diastolic or diastolic and systolic Ca2+ concentrations will be used to test the hypothesis that a defective sarcoplasmic reticulum Ca2+ release activates signaling mechanisms in the embryonic heart, and ensuing major alterations in signaling and Ca2+ handling proteins contribute to the rapid progression of cardiac hypertrophy in newborn mice. We will determine heart and cardiomyocyte function and morphology, and temporal changes in relative abundance and activity of signaling molecules by microarray, quantitative RT-PCR, immunoblot and enzymatic analysis. The functional significance of class II HDAC/MEF2 and calcineurin signaling will be probed using class II HDAC mutants, and by crossing mice impaired in CaM regulation of RyR2 with mice deficient in calcineurin A2, and with mice that carry the luciferase transgene driven by NFAT-dependent promoter. Ca2+ handling by wild type and mutant mice will be assessed using whole cell patch clamp techniques, Ca2+ imaging, cell homogenates and membrane preparations. PUBLIC HEALTH RELEVANCE The proposed research will make use of genetically modified mice with mutations in the calmodulin binding domain of the cardiac sarcoplasmic reticulum Ca2+ release channel to reveal new regulatory mechanisms in cardiac hypertrophy. Our studies will provide new approaches to minimize the risks of cardiac hypertrophy and heart failure, one of the most frequent causes of death in humans.

描述（由申请人提供）：我们提出的研究的目的是了解心脏CA2+释放通道（Ryanodine受体，RYR2）如何调节心脏功能。 RYR2是2200 kDa离子通道，响应于肌质网的作用电位而释放Ca2+离子。 RYR2离子通道由四个RYR2 560 kDa亚基组成，这些亚基结合了钙调蛋白（CAM）和四个小12.6 kDa FK506结合蛋白。我们提出的研究利用了基因修饰的小鼠在RYR2的CAM结合结构域中的突变，以阐明与心脏肥大相关的信号传导机制。表达RyR2突变形式的纯合小鼠（RYR2-W3587A/L3591D/F3603A或RYR2ADA），在舒张期和收缩期CA2+浓度下，CAM并未抑制这种症状，显示出在出生后1天的早期心脏肥大的迹象。与II类组蛋白脱乙酰基酶（HDAC）/肌细胞增强子因子2（MEF2）和钙调神经蛋白信号通路以及纯合突变小鼠在出生后的两周内死亡。 Genetically modified mice deficient in CaM regulation of RyR2 by CaM at diastolic or diastolic and systolic Ca2+ concentrations will be used to test the hypothesis that a defective sarcoplasmic reticulum Ca2+ release activates signaling mechanisms in the embryonic heart, and ensuing major alterations in signaling and Ca2+ handling proteins contribute to the rapid progression of cardiac hypertrophy in newborn mice.我们将通过微阵列，定量RT-PCR，免疫印迹和酶促分析来确定心脏和心肌细胞功能和形态，以及信号分子的相对丰度和活性的时间变化。 II类HDAC/MEF2和钙调神经蛋白信号的功能意义将使用II类HDAC突变体进行探测，并通过将RYR2在CAM调节中损害的小鼠与钙调神经磷酸酶A2缺乏小鼠的小鼠以及携带由NFAT依赖于NFAT依赖于NFAT依赖的发起人驱动的小鼠。将使用野生型和突变小鼠处理Ca2+处理，将使用全细胞贴片夹技术，Ca2+成像，细胞匀浆和膜制剂进行评估。公共卫生相关性拟议的研究将利用基因修饰的小鼠在心脏肌浆网的钙调蛋白结合结构域中具有突变的小鼠Ca2+释放通道，以揭示心脏肥大中的新调节机制。我们的研究将提供新的方法，以最大程度地减少心脏肥大和心力衰竭的风险，这是人类死亡最常见的原因之一。