Central GRK5 modulation of Angiotensin II receptor expression in heart failure

描述（由申请人提供）：本提案将解决以下具体目标：（1）确定正常条件下和慢性心力衰竭（CHF）期间中枢GRK 5在调节AT 1 R（血管紧张素II 1型受体）表达中的作用。 (2)确定CHF动物运动训练（ExT）后中枢AT 1 R和GRK 5的调节机制。 (3)确定的作用，胞浆和核GRK 5在转录调节AT 1 R的I？B和NF-？B。在特定目标1中，我们将通过冠状动脉结扎诱导CHF。GRK 5的过表达将通过慢病毒注射靶向RVLM或PVN。为了确定GRK 5敲低对AT 1 R表达和交感神经兴奋变化的影响，我们将利用市售GRK 5 KO小鼠和将注射到RVLM或PVN中的针对GRK 5的慢病毒包装siRNA。将在所有动物组中测量去甲肾上腺素（NE）、血浆NE和Ang II的尿排泄。将连续记录动脉压和心率，以获得交感神经兴奋的其他指数并确定动脉压力反射功能。在终末实验中，将在麻醉下直接记录交感神经活动。在所有动物中，心脏 将通过高频超声心动图连续评价功能。在特定目标2中，我们将在久坐或ExT的GRK 5 KO小鼠中诱导CHF，以及利用其中GRK 5已使用siRNA慢病毒在PVN或RVLM中沉默的CHF大鼠。ExT后，将以与特定目标1相似的方式评价交感神经和压力反射功能。我们将测试具体目标3在CATH。a神经元，利用过表达和沉默技术与GRK 5质粒和siRNA检查AT 1 R，I？B，NF-？B和GRK 5。我们还将进行平行研究，使用K215 R显性阴性GRK 5构建，以确定是否GRK 5/AT 1 R/I？B/NF-？B相互作用依赖于激酶。 公共卫生相关性：慢性心力衰竭（CHF）是美国死亡的主要原因之一（每年600，000例新诊断），其特征部分在于交感神经活性增加，部分由血管紧张素II 1型受体（AT 1 R）的上调介导。运动训练是CHF患者接受的治疗方法，可以提高生存率，减少并发症，并消除肌肉交感神经活动的增加。更好地了解CHF期间和ExT后AT 1 R的调节可能会导致开发治疗方法和更有效地治疗这种疾病。

DESCRIPTION (provided by applicant): This proposal will address the following specific aims: (1) Determine the role(s) of central GRK5 in the regulation of AT1R (Angiotensin II type 1 Receptor) expression under normal conditions and during Chronic Heart Failure (CHF). (2) Identify the mechanism(s) of modulation of central AT1R and GRK5 following Exercise Training (ExT) in CHF animals. (3) Determine the role of cytosolic and nuclear GRK5 in the transcriptional regulation of AT1R by I?B¿ and NF-?B. In Specific Aim 1 we will induce CHF by coronary artery ligation. Overexpression of GRK5 will be targeted to the RVLM or PVN by lentiviral injection. To determine the effects of GRK5 knockdown on AT1R expression and changes in sympatho-excitation, we will utilize both commercially available GRK5 KO mice and lentiviral packaged siRNA against GRK5 that will be injected into the RVLM or PVN. Urinary excretion of norepinephrine (NE), plasma NE and Ang II will be measured in all animal groups. Arterial pressure and heart rate will be continuously recorded in order to derive additional indices of sympatho-excitation and to determine arterial baroreflex function. Sympathetic nerve activity will be directly recorded under anesthesia in terminal experiments. In all animals cardiac function will be evaluated serially by high-frequency echocardiography. In Specific Aim 2, we will induce CHF in GRK5KO mice that are either sedentary or ExT as well as utilize CHF rats in which GRK5 has been silenced in the PVN or RVLM using siRNA lentivirus. Following ExT, sympathetic and baroreflex function will be evaluated in a similar fashion as in Specific Aim 1. We will test Specific Aim 3 in CATH.a neurons, utilizing both overexpression and silencing techniques with a GRK5 plasmid and siRNA to examine the subcellular localization of AT1R, I?B¿, NF-?B, and GRK5 following Ang II stimulation. We will also perform parallel studies using a K215R dominant negative GRK5 construct to determine if the GRK5/AT1R/I?B¿/NF-?B interaction is kinase-dependent. PUBLIC HEALTH RELEVANCE: Chronic Heart Failure (CHF) is one of the leading causes of death in the United States (600,000 new diagnoses each year), and is characterized in part by increased sympathetic nerve activity mediated in part by upregulation of the Angiotensin II type 1 Receptor (AT1R). Exercise training is an accepted therapy in patients with CHF and can increase survival, decrease complications, and abrogate increases in muscle sympathetic nerve activity. Gaining a greater understanding of the regulation of AT1R both during CHF and following ExT may lead to development of therapeutics and more effective treatment of this disease.