精氨酸加压素1A受体在心脏中的作用

Heart failure (HF) secondary to systolic dysfunction is a progressive disease mediated in large part by the release of neurohormonal signals including arginine vasopressin (AVP) and subsequent activation of G protein-coupled receptors (GPCR) and their downstream signaling pathways. AVP levels are elevated in patients with HF，in which increased AVP levels are associated with increased morbidity and mortality. Among three AVP receptors, V1A, V2 and V1B, V1A receptor is solely expressed in hearts and mediates cardiac hypertrophy through Gq protein. However, V1AR signaling also appears to have protective effects as we found that stimulation with AVP in H9c2 cardiac cells inhibits cell death through GPCR kinase (GRK)-dependent activation of extracellular-regulated kinase (ERK1/2). In addition, we observed that the V1AR appears to be tightly coupled or associated with β-adrenergic receptors (βARs) as AVP signaling significantly decreases the physiologic response to βAR agonists in cardiomyocytes. Moreover, V1AR stimulation results in enhanced recruitment of βarrestin and activated ERK1/2 to β1AR, which could promote protective signaling. Thus, we hypothesize that GRK-dependent V1AR signaling relays cardioprotective effects and that V1AR regulation of βAR signaling modulates cardiac function and survival. The goals of this application are to elucidate the mechanisms and importance of both GRK dependent V1AR signaling and V1AR-dependent regulation of βAR signaling in the heart. The specific aims are as follows: 1) Determine the impact of GRK-dependent V1AR signaling in cardiomyocytes; 2) Elucidate the impact of V1AR-mediated regulation of cardiomyocyte βAR signaling; 3) Determine the impact of GRK/βarr-dependent V1AR signaling and V1AR-mediated effects on βAR signaling in the regulation of cardiac function and survival under conditions of cardiac injury in vivo. Innovation: The mechanisms of V1AR signaling and their relevance to cardiac function and survival during cardiac stress will, for the first time, be studied using adult cardiomyocytes and in vivo models of HF. These studies will take advantage of unique transgenic animals, reagents and technology available in the investigators' laboratory. Significance: The proposed studies will distinguish the molecular mechanism(s) responsible for V1AR-mediated cardiomyopathy versus adaptive signaling and provide the scientific underpinning to direct the development of GRK/βarr-biased therapeutic agents to promote cardioprotective V1AR signaling. Completion of these goals will facilitate the rational development of novel biased ligands designed to simultaneously block deleterious G protein-dependent signaling and promote cardioprotective GRK-dependent signaling, and/or modulation of βAR signaling, for use in HF patients with elevated levels of AVP.

临床上心力衰竭是一种渐进性疾病，其进展取决于神经激素释放（如精氨酸加压素-AVP）及其激活的G蛋白偶联受体（GPCR）和下游的分子信号传导。心脏中AVP受体V1A通过激活Gαq蛋白介导心肌肥大。但近来我们发现精氨酸加压素刺激H9c2心肌细胞，经GPCR激酶（GRK）依赖途径抑制细胞死亡；V1A受体信号明显降低了βAR的心肌细胞反应，但加强了β1AR诱导的βarrestin聚集。本研究假说：Gαq蛋白非依赖的V1A受体信号经GRK依赖途径,介导V1A受体的抗心肌细胞肥大和凋亡信号；V1A受体通过调节βAR信号转导影响心肌细胞功能。本研究将应用成年心肌细胞培养，结合腺病毒转染方法及多种转基因小鼠实验，阐明GRK依赖的V1A受体信号机制及其调控的βAR信号对正常心脏和心肌损伤的作用。研究结果将指导临床心衰病人应用AVP抑制剂和β-阻滞剂，并为开发具有选择性信号转导的V1A受体配体(偏向性配体)。

临床上心力衰竭是一种渐进性疾病，其进展取决于神经激素释放（如精氨酸加压素-AVP）及其激活的G蛋白偶联受体（GPCR）和下游的分子信号传导。心脏中AVP受体V1A通过激活Gαq蛋白介导心肌肥大。但近来我们发现精氨酸加压素刺激H9c2心肌细胞，经GPCR激酶（GRK）依赖途径抑制细胞死亡及V1A受体信号明显降低了βAR的心肌细胞反应，但加强了β1AR诱导的βarrestin聚集。因此，本研究在目前的研究基础上明确：（1）Gαq蛋白非依赖的V1A受体信号经GRK依赖途径,是否介导V1A受体的抗心肌细胞肥大和凋亡信号及成纤维细胞增殖作用；（2）V1A受体通过调节βAR信号转导影响心肌细胞功能。本项目研究主要集中在心脏V1A受体的GRK2 途径对心脏细胞的作用，即主要研究心脏V1A受体介导的GRK2途径在成纤维细胞增殖、炎症及血管平滑肌细胞增殖等作用; 同时以新生大鼠心肌细胞培养模型， 观察V1A受体激动时对β1AR引起的细胞死亡后肥大的影响。研究应用成年心肌细胞培养，结合腺病毒转染方法及多种转基因小鼠实验，阐明GRK2依赖的V1A受体信号机制在心脏炎症反应和心脏成纤维细胞增殖及血管平滑肌细胞增殖中作用。主要有以下主要结果： （1）GRK2/β-arrestin介导精氨酸加压素诱导的心脏成纤维细胞增殖 首次发现心脏成纤维细胞中只有V1A受体亚型存在, 并且ERK1/2信号分子为GRK2/β-arrestin靶向分子， 与细胞增殖反应有关； （2）GRK2介导精氨酸加压素经NF-kB途径引起的大鼠心脏成纤维细胞的IL-6表达 研究不仅首次发现GRK2与精氨酸加压素调节炎症分子有关， 还首次发现GRK2调控NF-kB的新模式; （3）GRK2-EFGR-ERK1/2途径介导精氨酸加压素经引起的血管平滑肌的增殖 研究首次发现精氨酸加压素通过V1A受体的GRK2偏向性信号分子GRK2介导EGFR活化（transaction）途径在血管平滑肌增殖中作用；（4）V1A 受体经beta-arretin 途径抑制β1AR引起的心肌细胞死亡和肥大。 研究结果将指导临床心衰病人应用AVP抑制剂和GRK2阻滞剂，并为开发具有选择性信号转导的V1A受体配体(偏向性配体)提供实验依据。