Unique Domain Features of GRK2 and Roles in Cardiovascular Disease

GRK2 的独特结构域特征及其在心血管疾病中的作用

• 批准号: 9899299
• 负责人: Sarah Marie Bass
• 金额: $ 24.9万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899299
• 关键词: ADRBK1 gene; Acute; Agonist; American; Atrial Fibrillation; Award; Binding; Binding Proteins; Binding Sites; Cardiac; Cardiac development; Cardiovascular Diseases; Cardiovascular system; Cause of Death; Cell Surface Receptors; Cellular biology; Chronic; Complex; Coronary artery; Data; Development; Disease; Disease Progression; Elements; Evaluation; Faculty; G protein coupled receptor kinase; G-Protein-Coupled Receptors; Gene Transduction Agent; Goals; Heart; Heart Diseases; Heart Hypertrophy; Heart Injuries; Heart failure; Human; Hypertension; Hypertrophy; Immunohistochemistry; In Vitro; Infarction; Injury; Intervention; Investigation; Lead; Left; Left Ventricular Remodeling; Length; Ligation; Link; Measures; Mediating; Modeling; Molecular; Mus; Muscle Cells; Myocardial Infarction; N-terminal; Pathway interactions; Patients; Peptides; Pharmacologic Substance; Phase; Phosphorylation; Physiological; Physiology; Play; Positioning Attribute; Proteins; Proteomics; RGS Domain; Regulation; Research; Resources; Role; Signal Pathway; Signal Transduction; Structure; Techniques; Testing; Therapeutic; Therapeutic Intervention; Time; Training; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; Up-Regulation; Ventricular; Voltage-Gated Potassium Channel; Work; cardiogenesis; career; comorbidity; constriction; experimental study; functional restoration; gain of function; gene therapy; heart function; heart preservation; immunocytochemistry; improved; in vivo; in vivo evaluation; insight; ischemic injury; mouse model; new therapeutic target; novel; pressure; prevent; protein protein interaction; protein transport; receptor; reduce symptoms; research facility; response; tenure track; therapeutic development; therapeutic target; tool; trafficking; vector

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.

项目概要/摘要 在过去的十年里，我的科学生涯一直致力于转化心血管研究。我的 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药物干预如何改变这些。加盟目标 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 在体内研究的背景下取得进展，更加关注人类的治疗干预 心力衰竭（HF）。当前提案生成的初步数据表明，氨基（N）端 GRK2 的 RGS（G 蛋白信号传导调节器）结构域（aa 45-185，βARKrgs）和较短的 N 端 GRK2 肽（氨基酸 45-185，βARKnt）在肌细胞中表达时可以改变心脏生理学。值得注意的是， 这两种肽似乎都能阻止小鼠压力超负荷后的心力衰竭进展，但具有不同的作用 对初始肥大反应的影响。该提案的 K99 部分将重点关注 βARKrgs 是否 βARKnt 可以在治疗上逆转心脏损伤后的左心室 (LV) 重塑。这些 研究将从评估 βARKrgs 或 βARKnt 基因疗法逆转疾病的体内功效开始 急性适应性肥大或在慢性压力超负荷期间恢复功能。除此之外，我还会继续 在高二和博士的指导下练习小鼠心肌梗死（MI）模型。 R00期间 在这个提案的阶段，我将使用心脏受限的转基因 βARKrgs 和 βARKnt 小鼠以及我的新- 开发了基因治疗载体来确定这些肽是否可以预防心肌梗死后的不良重塑。 在 K99 阶段，我还将使用蛋白质组学方法来识别 βARKrgs 的特异性结合伴侣 和 βARKnt 体内，与全长 GRK2 相比，以及这些结合相互作用在 心脏损伤或激动剂刺激。对于这些研究，我将与该中心主任 Salim Merali 博士密切合作。 坦普尔蛋白质组学研究设施，以获得宝贵的见解和正确执行和培训 蛋白质组分析的评估。在这些研究中，βARKrgs 和 βARKnt 将作为强大的工具来剖析 GRK2 N 末端内负责蛋白质相互作用的特定结构域及其发挥的作用 心血管细胞信号传导的调节。该项目中发现的新型蛋白质相互作用将 为独立研究提供新途径。 R00阶段的重点将是缩小范围并追求 代表心脏信号传导关键要素或潜在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 独立之路提供支持 K99/R00 奖项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，以实现获得终身教职的总体目标。