DDAH1 effects on the development of congestive heart failure

描述(由申请人提供)： DDAH1在充血性心力衰竭发生发展中的作用内皮型一氧化氮合酶(ENOS)产生的一氧化氮(NO)在维持血管内皮细胞功能和保护心脏免受不利的心室重构方面具有重要作用。内源性一氧化氮合酶抑制物不对称二甲基精氨酸和L精氨酸是高血压、充血性心力衰竭和动脉粥样硬化等心血管疾病的主要独立危险因素。这些内源性一氧化氮合酶抑制剂与L精氨酸竞争抑制eNOS产生NO。ADMA和L-NMMA主要通过二甲精氨酸二甲氨基水解酶代谢成L-瓜氨酸而被清除。DDAH1和DDAH2由两个不同的基因编码。我们已经产生的初步数据表明，DDAH1而不是DDAH2在肾和脑等组织中降解一氧化氮合酶抑制剂，从而调节这些组织中的NO生物利用度方面发挥着关键作用。在心脏中，我们发现DDAH1在冠状动脉内皮细胞和心肌细胞的肌膜下都有表达。为了解决DDAH1在调节内源性一氧化氮合酶抑制物、NO生物利用度和心血管功能方面的细胞特异性作用，我们培育了三个新的组织特异性DDAH1 KO小鼠品系。利用这些新菌株，我们提出了一些研究，以确定DDAH1的心脏保护作用是否存在于心肌细胞或冠状动脉内皮细胞中表达的DDAH1。本研究的具体目标包括：(I)利用内皮特异性和全局性DDAH1基因缺陷小鼠，研究总DDAH1及其在血管内皮细胞表达的DDAH1在降解心肌ADMA和L-NMMA中的作用；(Ii)确定表达于心肌细胞的DDAH1对慢性收缩超负荷所致心力衰竭的心脏保护作用；以及(Iii)阐明血管内皮细胞表达的DDAH1对慢性收缩超负荷所致心力衰竭的保护作用。我们将检测不同KO小鼠品系中NO生成和ROS生成的变化。我们实验室培育的这些独特的组织特异性DDAH1 KO小鼠将使我们能够阐明DDAH1的细胞类型特异性作用，以及DDAH1保护超负荷心脏的分子机制。 公共卫生相关性： 心血管疾病被列为美国头号杀手，每年导致近100万人死亡。众所周知，一氧化氮(NO)对心脏具有保护作用。内源性一氧化氮合酶抑制剂ADMA和L-NMMA的积累与心源性死亡增加和各种心血管疾病的发展有关，如高血压、冠心病、动脉粥样硬化和充血性心力衰竭(CHF)。二甲基精氨酸二甲氨基水解酶(DDAH)降解二甲基精氨酸二甲酯和L-N甲基甲基丙烯酸甲酯。然而，我们对这一一氧化氮合酶抑制系统的生理和病理的了解非常有限。例如，虽然DDAH被报道通过降解ADMA和L-NMMA来增加NO的生物利用度，但DDAH1与DDAH2在体内调节NO生物利用度的作用尚不清楚。此外，目前尚不清楚这些内源性一氧化氮合酶抑制剂的长期积累是否会直接导致或加剧心血管疾病。在这项申请中提出的研究将使用我们实验室产生的独特的组织特异性KO小鼠来阐明DDAH的异构体和细胞类型特异性的作用。需要检验的中心假设是：(I)DDAH1(而不是DDAH2)是心血管系统中ADMA和LNMMA降解的必需或唯一的酶，(Ii)内皮DDAH1的缺失将导致内源性NOS抑制物的积聚和全身性高血压，以及(Iii)DDAH1的缺失将通过降低NO的生物利用度而加剧超负荷心脏的CHF发展。我们有具体的初步数据来支持这些假设。我们将研究ADMA和L-NMMA的慢性蓄积对心肌NO生物利用度和一氧化氮合酶衍生的ROS产生的影响。最后，我们将确定血管内皮细胞或心肌细胞中DDAH1的缺失是否会损害心脏适应小鼠横动脉收缩所产生的慢性压力超负荷的能力。这些研究将为DDAH1如何调节NO和NOS来源的ROS的产生提供新的知识，并证明DDAH1在心肌细胞或血管内皮细胞中的异常调节是否有助于CHF的发生。

DESCRIPTION (provided by applicant): DDAH1 effects on the development of congestive heart failure Abstract Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is important in maintaining vascular endothelial function and in protecting the heart from adverse ventricular remodeling. Accumulation of the endogenous NOS inhibitors asymmetric dimethyl arginine (ADMA) and Ng-monomethyl-L-arginine (L-NMMA) is a major independent risk factor for cardiovascular diseases including hypertension, congestive heart failure and atherosclerosis. These endogenous NOS inhibitors compete with L-arginine to inhibit NO production by eNOS. ADMA and L-NMMA are eliminated principally by metabolism to L-citrulline by dimethylarginine dimethylaminohydrolase (DDAH). DDAH1 and DDAH2 are encoded by two different genes. We have generated preliminary data indicating that DDAH1 rather than DDAH2 plays the essential role in degrading the NOS inhibitors in tissues such as kidney and brain and thereby regulating NO bioavailability in these tissues. In the heart we find DDAH1 expressed both in coronary endothelium and under the sarcolemma of cardiac myocytes. To address the cell specific role of DDAH1 in regulating endogenous NOS inhibitors, NO bioavailability and cardiovascular function, we have generated three novel tissue specific DDAH1 KO mouse strains. Using these new strains, we propose studies to determine whether the cardioprotective effect of DDAH1 resides in DDAH1 expressed in the cardiac myocytes or in the coronary endothelium. Specific aims will be addressed: (i) Determine the role of total-DDAH1, and DDAH1 expressed in the vascular endothelium in degrading myocardial ADMA and L-NMMA using endothelial specific and global DDAH1 gene deficient mice; (ii) Determine the cardiac protective effect from DDAH1 expressed in the cardiomyocytes on the development of heart failure produced by chronic systolic overload; and (iii) elucidate the protective effect from DDAH1 expressed in the vascular endothelium on the development of heart failure after chronic systolic overload. We will examine both changes in NO production and ROS generation in the different KO mouse strains. These unique tissue specific DDAH1 KO mice generated in our laboratory will allow us to elucidate cell type specific actions of DDAH1, as well as molecular mechanisms by which DDAH1 protects the overloaded heart. PUBLIC HEALTH RELEVANCE: Cardiovascular disease ranks as America's No. 1 killer that accounts for nearly one million deaths each year. Nitric oxide (NO) is known to exert protective effects on the heart. Accumulation of the endogenous nitric oxide synthase (NOS) inhibitors ADMA and L-NMMA is associated with increased cardiac death and the development of various cardiovascular diseases such as hypertension, coronary disease, atherosclerosis and congestive heart failure (CHF). ADMA and L-NMMA are degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). However, our understanding of the physiology and pathology of this NOS inhibitor system is very limited. For example, although DDAH is reported to increase NO bioavailability by degradation of ADMA and L-NMMA, the in vivo role of DDAH1 vs. DDAH2 in regulating NO bioavailability is not clear. In addition, it is not known whether chronic accumulation of these endogenous NOS inhibitors can directly cause or exacerbate cardiovascular disease. The studies proposed in this application will use unique tissue specific KO mice generated in our laboratory to elucidate both isoform- and cell type specific actions of DDAH. The central hypotheses to be tested are that (i) DDAH1 (not DDAH2) is the essential or sole enzyme responsible for degradation of ADMA and LNMMA in cardiovascular system, (ii) deletion of endothelial DDAH1 will cause accumulation of the endogenous NOS inhibitors and systemic hypertension, and (iii) deletion of DDAH1 will exacerbate the development of CHF in the overloaded heart by decreasing NO bioavailability. We have concrete preliminary data to support these hypotheses. We will examine the influence of chronic accumulation of ADMA and L-NMMA on myocardial NO-bioavailability and on NOS-derived ROS generation. Finally, we will determine whether deletion of DDAH1 in endothelium or in cardiac myocytes impairs the ability of the heart to adapt to chronic pressure overload produced by transverse aortic constriction in mice. These studies will provide new knowledge regarding how DDAH1 acts to regulate production of NO and NOS-derived ROS, and demonstrate whether dysregulation of DDAH1 in either cardiac myocytes or endothelium can contribute to the development of CHF.