Implications of Endothelial-Myocyte Uncoupling in Cardiac Arrhythmia

描述（由申请人提供）：高同型半胱氨酸（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。它增加了iNOS，激活了基质金属蛋白酶（MMP），破坏了连接蛋白43，增加了胶原蛋白/弹性蛋白的比例。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，减弱一氧化氮从内皮细胞到肌细胞（E-M）的运输，导致E-M解耦。该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，阻断NMDA- r1可减少SCO。该建议的中心假设是Hcy增加iNOS， mtNOS活性，超氧化物水平，金属蛋白酶活性，破坏连接蛋白43，加剧内皮-肌细胞解偶联，并通过激活NMDA-R1诱导心力衰竭。具体目的1：确定Hey是否通过增加iNOS并通过作为NMDA-R1的激动剂使eNOS和nNOS无效而加剧心力衰竭和内皮-肌细胞解耦。将CBS杂合子（-/+）敲除（CBSKO）小鼠与iNOS纯合子（-/-）敲除（iNOSKO）小鼠杂交，产生野生型（WT）、CBSKO、iNOSKO和CBS/iNOS（-/+; -/-）双敲除（doubleKO）。在这些小鼠中，慢性容量超负荷心力衰竭将由主动脉-静脉（AV）瘘引起。NMDA-R1将被二唑西平（MK-801）阻断。内皮-肌细胞耦合将在心脏环中确定。测量NMDA-R1、iNOS、nNOS和eNOS的LV水平。具体目的2：确定Hey是否通过诱导NMDA-R1增加心力衰竭患者的MMP-2， -9, -13, ADAM-12，降低TIMP-4，并降解连接蛋白43。MMP和TIMP活性将分别通过创新的2-酶谱（MMP功能蛋白质组学）和反向酶谱测定。连接蛋白43、胶原蛋白和弹性蛋白的降解将通过Western分析来测量。特异性目的#3：确定Hey是否通过激活NMDA-R1降低心衰患者左室线粒体硫氧还蛋白、过氧化物还蛋白和SOD，并增加NADH氧化酶和mtNOS活性。在心脏中，将对硫氧还蛋白、过氧化物还蛋白、SOD和NADH氧化酶进行原位标记。这些研究将描述心律失常和心力衰竭中hcy依赖性内皮-肌细胞解耦的机制，并将对心源性猝死的治疗产生影响。

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.