Central GRK5 modulation of Angiotensin II receptor expression in heart failure

描述（由申请人提供）：该提案将解决以下具体目标：(1)确定中央GRK5在正常情况下和慢性心力衰竭（CHF）期间调节AT1R（血管紧张素II型1受体）表达中的作用。(2)确定运动训练（ExT）后CHF动物中枢AT1R和GRK5调节的机制。(3)确定胞质和细胞核GRK5在I？B¿和NF-？在专项目的1中，我们将通过冠状动脉结扎诱导CHF。GRK5过表达将通过慢病毒注射靶向RVLM或PVN。为了确定GRK5敲低对AT1R表达和交感神经兴奋变化的影响，我们将利用市售GRK5 KO小鼠和慢病毒包装的靶向GRK5的siRNA，将其注射到RVLM或PVN中。测定各组动物尿中去甲肾上腺素（NE）、血浆NE和Ang II的排泄量。动脉压和心率将被连续记录，以得出交感神经兴奋的额外指标，并确定动脉压力反射功能。在麻醉状态下直接记录交感神经的活动。在所有心脏动物中

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.