Implications of Endothelial-Myocyte Uncoupling in Cardiac Arrhythmia

描述(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)，与心律失常和心脏性猝死(SCO)有关。HY增加iNOS，激活基质金属蛋白酶，破坏连接蛋白-43，增加胶原/弹性蛋白比率。缝隙连接蛋白-43的破坏和胶原蛋白的积聚(纤维化)阻断了心脏传导，减弱了一氧化氮从内皮到心肌细胞(E-M)的运输，导致E-M解偶联。这一建议的新颖之处在于，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，而阻断NMDA-R1可降低SCO。这一设想的中心假设是Hcy通过激活NMDA-R1，增加iNOS、mtNOS活性、超氧化物歧化水平、金属蛋白酶活性，破坏连接蛋白-43，加剧内皮-心肌细胞解偶联，从而导致心力衰竭。具体目的#1：确定嘿是否通过增加iNOS而使无效的eNOS和nNOS成为NMDA-R1的激动剂而加剧心力衰竭和血管内皮细胞-心肌细胞解偶联。CBS杂合子(-/)基因敲除(CBSKO)小鼠将与iNOS纯合子(-/-)基因敲除(INOSKO)小鼠杂交，产生野生型(WT)、CBSKO、iNOSKO和CBS/iNOS(-/；-/-)双基因敲除(DoubleKO)。在这些小鼠中，主动脉-下腔静脉(AV)瘘将造成慢性容量超负荷性心力衰竭。地佐西平(MK-801)可阻断NMDA-R1。将在心环中确定内皮细胞-心肌细胞偶联。测定左心室NMDA-R1、iNOS、nNOS和eNOS水平。特定目的#2：确定Hey是否通过诱导NMDA-R1增加心力衰竭时基质金属蛋白酶-2、-9、-13、ADAM-12、降低TIMP-4和降解连接蛋白-43。基质金属蛋白酶和组织基质金属蛋白酶活性将分别通过创新的2-酶图谱(基质金属蛋白酶功能蛋白质组学)和反向酶图谱进行测定。Western分析检测细胞间隙连接蛋白-43、胶原和弹性蛋白的降解情况。具体目的#3：确定HY是否通过激活NMDA-R1降低心力衰竭大鼠左室心肌线粒体硫氧还蛋白、过氧化还蛋白和超氧化物歧化酶的活性，增加NADH氧化酶和线粒体一氧化氮合酶的活性。在心脏，硫氧还蛋白、过氧化还蛋白、超氧化物歧化酶和NADH氧化酶将进行原位标记。这些研究将阐明Hcy依赖的内皮-心肌细胞解偶联在心律失常和心力衰竭中的机制，并将对心脏性猝死具有治疗意义。

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.