Unique Domain Features of GRK2 and Roles in Cardiovascular Disease

项目总结/摘要 在过去的10年里，我的科学生涯一直致力于转化心血管研究。我 博士研究调查了电压门控钾通道Kv1.5作为潜在治疗药物的作用 目标是房颤。这些研究有很强的细胞生物学重点，确定了潜在的机制， 渠道贩运和监管，以及如何通过药物干预改变这些。加入的目标 我在科赫实验室进行博士后研究是为了拓宽我对心血管疾病的理解 在体内研究背景下的进展，更侧重于人类的治疗干预 心力衰竭（HF）。针对当前提案生成的初步数据显示，氨基（N）-末端 GRK 2的RGS（Regulator of G-protein Signaling）结构域（aa 45-185，βARKrgs）和较短的N-末端 GRK 2（aa 45-185，βARKnt）肽在心肌细胞中表达时可改变心脏生理学。值得注意的是， 这两种肽似乎都能阻止小鼠在压力超负荷后的HF进展， 对初始肥大反应的影响。本提案的K99部分将重点关注βARKrgs 而βARKnt可以在治疗上逆转心脏损伤后的左心室（LV）重构。这些 研究将开始评估βARKrgs或βARKnt基因治疗逆转 急性适应性肥大或在慢性压力超负荷期间恢复功能。此外，我将继续 在高二和博士的指导下进行小鼠心肌梗死模型的制作。在R 00期间 在这个提议的第一阶段，我将使用心脏限制性转基因βARKrgs和βARKnt小鼠，以及我的新- 开发了基因治疗载体，以确定这些肽是否能预防MI后的不良重塑。 在K99期间，I期还将使用蛋白质组学方法鉴定βARKrgs的特异性结合伴侣 与全长GRK 2相比，在体内，这些结合相互作用是否在 心脏损伤或激动剂刺激后。对于这些研究，我将与Salim Merali博士密切合作， 在坦普尔的蛋白质组学研究设施，以获得宝贵的见解和培训，在适当的执行和 蛋白质组学分析的评价。在这些研究中，βARKrgs和βARKnt将作为剖析 GRK 2 N端负责蛋白质相互作用的特定结构域及其作用 在调节心血管细胞信号方面的作用。该项目中发现的新蛋白质相互作用将 为独立研究提供了新的途径。R 00阶段的重点是缩小范围， 代表心脏信号传导的关键元件或潜在的治疗靶点的蛋白质结合伴侣， 改善疾病中的心脏结构和功能。通过NIH途径支持独立 K99/R 00奖将为实现这些重要的研究目标提供必要的时间和资源， 并继续我的个人发展，以实现我获得终身教职的总体目标。

PROJECT SUMMARY/ABSTRACT For the past 10 years my scientific career has been devoted to translational cardiovascular research. My doctoral studies investigated the role of the voltage-gated potassium channel Kv1.5 as a potential therapeutic target atrial fibrillation. These studies had a strong cell biology focus, determining the mechanisms underlying channel trafficking and regulation and how these were altered by pharmaceutical intervention. A goal in joining the Koch lab for my postdoctoral studies was to broaden my understanding of cardiovascular disease progression within the context of in vivo studies, with a greater focus on therapeutic interventions for human heart failure (HF). Preliminary data generated for the current proposal shows that both the amino(N)-terminal RGS (Regulator of G-protein Signaling) domain of GRK2 (aa 45-185, βARKrgs) and a shorter N-terminal peptide of GRK2 (aa 45-185, βARKnt) can alter cardiac physiology when expressed in myocytes. Of note, these two peptides both appear to halt HF progression in mice after pressure-overload but have differential effects on the initial hypertrophic response. The K99 portion of this proposal will focus on whether βARKrgs and βARKnt can act therapeutically to reverse left-ventricular (LV) remodeling after cardiac injury. These studies will begin with an evaluation of the in vivo efficacy of βARKrgs or βARKnt gene-therapy to reverse adaptive hypertrophy acutely or restore function during chronic pressure overload. In addition, I will continue to practice the murine myocardial infarction (MI) model under the guidance of Dr. Erhe Gao. During the R00 phase of this proposal I will use the cardiac-restricted transgenic βARKrgs and βARKnt mice and my newly- developed gene therapy vectors to determine whether these peptides prevent adverse remodeling post-MI. During the K99 phase I will also use proteomic approaches to identify specific binding partners for βARKrgs and βARKnt in vivo, compared to full-length GRK2, and whether these binding interactions are altered after cardiac injury or upon agonist stimulation. For these studies I will work closely with Dr. Salim Merali, Director of the Proteomics Research Facility at Temple, to gain invaluable insight and training in the proper execution and evaluation of proteomic analysis. In these studies βARKrgs and βARKnt will serve as powerful tools to dissect the specific domains within the N-terminus of GRK2 responsible for protein interactions and the role they play in the regulation of cardiovascular cell signaling. Novel protein interactions discovered in this project will provide new avenues for independent research. A focus of the R00 phase will be to narrow down and pursue the protein binding partners that represent key elements of cardiac signaling or potential therapeutic targets for improving cardiac structure and function in disease. Support through the NIH Pathway to Independent K99/R00 award would provide the necessary time and resources for achieving these important research goals, and continuing my personal development toward my overall goal of obtaining a tenure-track faculty position.