G蛋白偶联受体激酶2在精氨酸加压素诱导心衰中的作用及机制

Circulating and myocardium levels of arginine vasopressin (AVP), interleukin 6 (IL-6) and inflammasome-activated interleukin 1beta (IL-1beta) are elevated during heart failure and cardiac ischemia. Role of G-protein coupled receptor kinase (GRK2) overdriving by GPCR (G-protein coupled receptor) stimulation with high level of hormone in heat failure in regulation of cardiac non-infective inflammation and activation of inflammasome are unclear. Our preliminary experimental results show that perfusion of AVP increased the levels of IL-6 and IL-1beta mRNA in myocardium, caused myocardium damage and reduced cardiac contraction---effects that are mediated by GRK2-regualted NF-kB signaling pathway. Thus, we hypothesize that AVP enhances NF-kB to activate inflammasome resulting in heart failure by V1A/GRK2 biased signaling pathway. Using the cutting-edge techniques in the cultured cardiac myocytes and fibroblasts in vitro and transgenic mice in vivo, present study will determine the roles of GRK2 and its targeting NF-kB in AVP-induced secretion of inflammatory factors and activation of inflammasome by manipulating GRK2 activity with either small molecule inhibitors of GRK2 or cardiac myocyte-specific GRK2 knockout mice. The study will determine that: (1) AVP induces the expression of inflammatory factors and activation of inflammasome in cardiac myosytes and fibroblasts; (2) Cardiac myocyte specific GRK2-regulated NF-kB mediates the AVP-induced cardiac non-infective inflammation resulting in heart failure; （3） NF-kB mediates the AVP-induced cardiac non-infective inflammation resulting in heart failure by V1A/GRK2 signal dependent pathway ; (4) Both GRK2 inhibitor and V1A receptor selective antagonist protects the failure heart induced by AVP-perfusion and cardiac ischemia/reperfusion. The results will provide an experimental basis for GRK2 as a drug target for treatment of cardiac inflammatory and failing heart.

心衰或心肌缺血时血液和心肌组织精氨酸加压素（AVP）和白细胞介素-6（IL-6）及由炎症小体激活的白细胞介素-1beta(IL-1beta)升高。心衰时活性上调的G蛋白偶联受体激酶介导（GRK2）的偏向性信号途径在炎症引起的心衰中作用并不清楚。我们前期研究发现AVP灌注导致心脏IL-6和IL-1beta表达升高、心脏损伤和收缩功能下降，且GRK2经NF-kB p65途径介导AVP的V1A受体引起的大鼠心脏细胞的IL-6和IL-1beta的表达。因此我们的科学假说是AVP通过V1A/GRK2偏向信号途径活化NF-kB激活炎症小体诱导心衰。本项目将采用心肌细胞选择性GRK2基因敲除小鼠等多学科研究手段以明确：（1）AVP介导心脏细胞炎症因子表达和炎症小体活化；（2）心肌细胞特异性GRK2介导AVP的作用；（3）GRK2调节NF-kB活性机制；（4）GRK2抑制剂和V1A拮抗剂对心脏的保护作用。

业已表明，心力衰竭时神经内分泌激素功能紊乱及其心脏中G蛋白偶联受体（GPCR），包括G蛋白偶联受体激酶2(GRK2)功能的改变。目前，临床上针对GPCR 如Beta受体阻断剂和血管紧张素受体II阻断剂的广泛使用，提示神经内分泌激素功能紊乱学说在心力衰竭形成机制中有一定的科学依据。但任然有近一半心力衰竭病人用该类阻断剂效果不明显，因此有必要探索新的机制和途径， 提高心力衰竭的治疗效果。近来在心脏GPCR的信号转导中， BetaArrestin、GRK2途径介导多种心脏GPCR的生物学作用。 本项目在我们课题组前期研究的基础上，主要研究GRK2和BetaArrestin在精氨酸加压素（AVP）诱导心衰中的作用。 根据研究项目的科学假设：AVP通过V1A/GRK2偏向信号途径活化NF-kB激活炎症反应诱导心衰， 即心肌缺血时心脏组织AVP的升高，可能经心脏细胞V1A受体激活GRK2和Betaarretin信号分子， 经NF-kB途径调节细胞炎症因子分泌和炎症小体活化，导致心脏细胞的非感染性炎症反应，引起心肌心力衰竭。本项目采用采用GRK2小分子抑制剂和心肌GRK2和BetaArrestin基因敲除小鼠以分别调控GRK2h和BetaArrestin活性，采用qPCR、酶联免疫吸附测定（ELISA）、心脏功能测定、免疫组织化学、蛋白免疫印迹等实验技术，进一步明确：（1）AVP引起的炎症因子表达、炎症小体活化等在心脏损伤中作用；（2）GRK2 阻断剂和V1A受体选择性拮抗剂的阻断AVP的心脏炎症反应。本项目进一步研究Betaarrestin 是否与精氨酸加压素（AVP）经V1A受体从而介导心脏的炎症反应。项目研究明确：（1） AVP诱导心脏组织NLRP3炎症体和IL-1β的表达；（2）PDTC 通过阻断NF-κB p65 通路抑制AVP诱导的ARCFs；（3）PDTC通过阻断NF-κB p65通路抑制AVP诱导的ARCFsNLRP3炎症体和IL-1β的表达；（4）AVP诱导的心脏NLRP3炎症体的表达依赖于β-arrestin 2; (5)AVP诱导的ARCFs和小鼠NLRP3炎症体的表达依赖于β-arrestin 2。研究结果，阐明了GRK2和BetaArrestin在心力衰竭时血浆AVP水平升高时调节心脏炎症反应的新机制， 为临床心力衰竭的治疗提供新靶点。