Implications of Endothelial-Myocyte Uncoupling in Cardiac Arrhythmia

内皮-肌细胞解偶联对心律失常的影响

• 批准号: 7408062
• 负责人: Suresh C. Tyagi
• 金额: $ 37万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-18 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7408062
• 关键词: 3-deazaadenosine; Abbreviations; Acetylcholine; Address; Adenosine Diphosphate; Agonist; Alteplase; Aorta; Arginine; Arrhythmia; Attenuated; Basement membrane; Bradykinin; Breeding; Cardiac; Chronic; Collagen; Connexin 43; Coupling; Crossbreeding; Cystathionine; Disintegrins; Disruption; Dose; EKG QRS Complex; Echocardiography; Eicosatrienoic Acid; Elastin; Electrocardiogram; Endothelial Cells; Endothelin-1; Endothelium; Environment; Extracellular Matrix; Failure; Fibrosis; Fistula; Fluorescent Dyes; Gelatinase A; Goals; Heart; Heart Rate; Heart failure; Heterozygote; Homocysteine; Homocystine; Homozygote; Hydrolase; Hydroxyeicosatetraenoic Acids; Hyperhomocysteinemia; Image; In Situ; Knock-out; Label; Left ventricular structure; Lipopolysaccharides; Localized; MTHFR gene; Matrix Metalloproteinases; Measures; Mediating; Metalloproteases; Methionine; Methylenetetrahydrofolate reductase (NADPH); Mitochondria; Mus; Muscle Cells; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NADH oxidase; NADP; NOS1 protein, human; Neurons; Neurotransmitter Receptor; Niacinamide; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Norepinephrine; Oxidation-Reduction; Oxides; Pattern; Polymerase Chain Reaction; Preparation; Production; Proteomics; Radio; Reactive Nitrogen Species; Reactive Oxygen Species; Relaxation; Research Personnel; Shunt Device; Superoxide Dismutase; Superoxides; Tail; Telemetry; Therapeutic; Thioredoxin; Time; Tissue Inhibitor of Metalloproteinases; Veins; Ventricular Fibrillation; Ventricular Tachycardia; Western Blotting; analog; dizocilpine; human NOS3 protein; in vivo; innovation; membrane-type matrix metalloproteinase; peroxiredoxin; programs; relating to nervous system; relaxing factor; response; sudden cardiac death; tetrahydrobiopterin; tissue inhibitor of metalloproteinase 4; tumor necrosis factor receptor superfamily, member 10b protein, mouse

DESCRIPTION (provided by applicant): Elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with cardiac arrhythmia and sudden cardiac death (SCO). Hey increases the iNOS, activates matrix metalloproteinase (MMP), disrupts connexin-43 and increases collagen/elastin ratio. The disruption of connexin-43 and accumulation of collagen (fibrosis) interrupts cardiac conduction and attenuate NO transport from endothelium to myocyte (E-M) causing E-M uncoupling. The novelty of this proposal is that Hcy behaves as an agonist to N-methyl-D-aspartate (NMDA, an excitatory neurotransmitter) receptor-1, and blockade of NMDA-R1 reduces SCO. The central hypothesis of this proposal is that Hcy increases iNOS, mtNOS activities, superoxide levels, metalloproteinase activity, disrupts connexin-43, exacerbates endothelial-myocyte uncoupling, and induces cardiac failure by activating NMDA-R1. Specific Aim #1: To determine whether Hey exacerbates heart failure and endothelial-myocyte uncoupling by increasing iNOS and rendering ineffective eNOS and nNOS by behaving as an agonist to NMDA-R1. CBS heterozygote (-/+) knockout (CBSKO) mice will be crossbred with iNOS homozygote (-/-) knockout (iNOSKO) mice, producing wild type (WT), CBSKO, iNOSKO and CBS/iNOS (-/+; -/-) double knockout (doubleKO). In these mice, chronic volume overload heart failure will be created by aorta-venacava (AV) fistula. NMDA-R1 will be blocked by dizocilpine (MK-801). The endothelial-myocyte coupling will be determined in cardiac rings. LV levels of NMDA-R1, iNOS, nNOS and eNOS will be measured. Specific Aim #2: To determine whether Hey increases MMP-2, -9, -13, ADAM-12, decreases TIMP-4, and degrades connexin-43 in heart failure by inducing NMDA-R1. MMP and TIMP activities will be measured by innovative 2-zymography (MMP functional proteomics) and reverse zymography, respectively. The degradation of connexin-43, collagen and elastin will be measured by Western analysis. Specific Aim #3: To determine whether Hey decreases LV mitochondrial thioredoxin, peroxiredoxin, and SOD, and increases NADH oxidase and mtNOS activity in heart failure by activating NMDA-R1. In hearts, in situ labeling will be performed for thioredoxin, peroxiredoxin, SOD, and NADH oxidase. These studies will delineate the mechanism of Hcy-dependent endothelial-myocyte uncoupling in cardiac arrhythmia and failure, and will have therapeutic ramifications for sudden cardiac death.

描述（由申请人提供）：同型半胱氨酸（Hcy）水平升高（称为高同型半胱氨酸血症（HHcy））与心律失常和心源性猝死（SCO）相关。 Hey 增加 iNOS、激活基质金属蛋白酶 (MMP)、破坏 connexin-43 并增加胶原蛋白/弹性蛋白比率。连接蛋白 43 的破坏和胶原蛋白的积累（纤维化）会中断心脏传导并减弱 NO 从内皮到肌细胞 (E-M) 的转运，从而导致 E-M 解偶联。该提议的新颖之处在于，Hcy 作为 N-甲基-D-天冬氨酸（NMDA，一种兴奋性神经递质）受体 1 的激动剂，并且阻断 NMDA-R1 可减少 SCO。该提议的中心假设是，Hcy 增加 iNOS、mtNOS 活性、超氧化物水平、金属蛋白酶活性，破坏 connexin-43，加剧内皮肌细胞解偶联，并通过激活 NMDA-R1 诱导心力衰竭。具体目标#1：确定 Hey 是否通过增加 iNOS 并通过充当 NMDA-R1 激动剂而使 eNOS 和 nNOS 无效而加剧心力衰竭和内皮肌细胞解偶联。 CBS 杂合子 (-/+) 敲除 (CBSKO) 小鼠将与 iNOS 纯合子 (-/-) 敲除 (iNOSKO) 小鼠杂交，产生野生型 (WT)、CBSKO、iNOSKO 和 CBS/iNOS (-/+; -/ -) 双淘汰赛（doubleKO）。在这些小鼠中，主动脉-腔静脉 (AV) 瘘将导致慢性容量超负荷心力衰竭。 NMDA-R1 将被地佐西平 (MK-801) 阻断。内皮-肌细胞耦合将在心脏环中确定。将测量 NMDA-R1、iNOS、nNOS 和 eNOS 的 LV 水平。具体目标 #2：确定 Hey 是否通过诱导 NMDA-R1 来增加心力衰竭中的 MMP-2、-9、-13、ADAM-12，减少 TIMP-4 并降解 connexin-43。 MMP 和 TIMP 活性将分别通过创新的 2-酶谱法（MMP 功能蛋白质组学）和反向酶谱法进行测量。连接蛋白 43、胶原蛋白和弹性蛋白的降解将通过 Western 分析进行测量。具体目标#3：确定 Hey 是否通过激活 NMDA-R1 来降低心力衰竭患者左心室线粒体硫氧还蛋白、过氧化氧还蛋白和 SOD，并增加 NADH 氧化酶和 mtNOS 活性。在心脏中，将对硫氧还蛋白、过氧化氧还蛋白、SOD 和 NADH 氧化酶进行原位标记。这些研究将阐明心律失常和心力衰竭中 Hcy 依赖性内皮肌细胞解偶联的机制，并对心源性猝死产生治疗影响。