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）：称为高硬同性半胱氨酸血症（HHCY）的同型半胱氨酸（HCY）水平升高与心律不齐和心脏猝死有关（SCO）。嘿，增加了iNOS，激活基质金属蛋白酶（MMP），破坏连接蛋白43并增加胶原蛋白/弹性蛋白比。连接蛋白43的破坏和胶原蛋白（纤维化）的积累会中断心脏传导，并减弱从内皮到肌细胞（E-M）的转运，从而导致E-M解偶联。该提议的新颖性是，HCY是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，而NMDA-R1的阻断减少了SCO。该提案的中心假设是HCY增加了iNOS，mTNOS活性，超氧化物水平，金属蛋白酶活性，破坏连接蛋白43，加剧内皮 - 膜细胞解偶联，并通过激活NMDA-R1来诱导心脏衰竭。具体目的1：确定Hey是否通过增加iNOS和呈现无效的eNOS和nNOS来加剧心力衰竭和内皮膜细胞的解偶，并通过表现为NMDA-R1的激动剂。 CBS杂合子（ - /+）基因敲除（CBSKO）小鼠将与iNOS纯合子（ - / - ）敲除（Inosko）小鼠杂交，产生野生型（WT），CBSKO，Inosko，Inosko和CBS/CBS/Inos（ - /+; - /+; - / - - - / - ）双基因敲除（ - double doubleko）。在这些小鼠中，慢性体积超负荷心力衰竭将由主动脉 - venacava（AV）瘘管产生。 NMDA-R1将被Dizocilpine（MK-801）阻止。内皮膜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。特定目标＃2：确定HEY是否会增加MMP-2，-9，-13，ADAM-12，通过诱导NMDA-R1来减少TIMP-4，并降低Connexin-43。 MMP和TIMP活性将分别通过创新的2酶学（MMP功能蛋白质组学）和反向Zymography进行测量。连接蛋白43，胶原蛋白和弹性蛋白的降解将通过西方分析来测量。具体目的＃3：确定HEY是否通过激活NMDA-R1来减少LV线粒体硫氧还蛋白，过氧蛋白和SOD，并增加心力衰竭的NADH氧化酶和mTNOS活性。在心脏中，将对硫氧还蛋白，过氧蛋白，SOD和NADH氧化酶进行原位标记。这些研究将描述心律不齐和衰竭中HCY依赖性内皮膜细胞的机制，并将对心脏猝死产生治疗后果。