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: 7760565
• 负责人: GERHARD W MEISSNER
• 金额: $ 37万
• 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/7760565
• 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标是了解心脏 Ca2+ 释放通道（兰尼碱受体，RyR2）如何调节心脏功能。 RyR2 是 2,200 kDa 的离子通道，可响应肌浆网的动作电位释放 Ca2+ 离子。 RyR2 离子通道由四个结合钙调蛋白 (CaM) 的 RyR2 560 kDa 亚基和四个小的 12.6 kDa FK506 结合蛋白组成。我们提出的研究利用 RyR2 CaM 结合域突变的转基因小鼠来阐明与心脏肥大相关的信号传导机制。表达 RyR2 突变体（RyR2-W3587A/L3591D/F3603A 或 RyR2ADA）的纯合子小鼠，在舒张期和收缩期 Ca2+ 浓度下不受 CaM 抑制，早在出生后 1 天就显示出心脏肥大的迹象。与II类组蛋白脱乙酰酶(HDAC)/肌细胞增强因子2(MEF2)和钙调磷酸酶信号通路相关的基因和蛋白上调，纯合突变小鼠在出生两周内死亡。转基因小鼠在舒张期或舒张期和收缩期 Ca2+ 浓度下缺乏 CaM 对 RyR2 的调节，将用于测试以下假设：有缺陷的肌浆网 Ca2+ 释放会激活胚胎心脏中的信号传导机制，并随后导致信号传导和 Ca2+ 处理发生重大变化蛋白质有助于新生小鼠心脏肥大的快速进展。我们将通过微阵列、定量 RT-PCR、免疫印迹和酶分析确定心脏和心肌细胞的功能和形态，以及信号分子相对丰度和活性的时间变化。将使用 II 类 HDAC 突变体，并通过将 RyR2 CaM 调节受损的小鼠与缺乏钙调神经磷酸酶 A2 的小鼠以及携带由 NFAT 驱动的荧光素酶转基因的小鼠杂交，来探讨 II 类 HDAC/MEF2 和钙调磷酸酶信号传导的功能意义。依赖启动子。将使用全细胞膜片钳技术、Ca2+成像、细胞匀浆和膜制剂来评估野生型和突变型小鼠的Ca2+处理。公共健康相关性拟议的研究将利用心脏肌浆网 Ca2+ 释放通道的钙调蛋白结合域发生突变的转基因小鼠来揭示心脏肥大的新调节机制。我们的研究将提供新的方法来最大限度地降低心脏肥大和心力衰竭的风险，这是人类最常见的死亡原因之一。