Project 1: Targeting GRK5 in Cardiac Injury and Repair

项目1-摘要 临床综合征心力衰竭(HF)仍然是世界范围内的一个主要健康问题。这是一个终点 在心脏损伤/应激之后，这通常发生在心脏肥厚和不利的心室重构之前。 在过去的二十年里，我们的实验室一直在研究HF的发展，重点是G蛋白的作用- 偶联受体(GPCR)信号传导。特别是，我们感兴趣的是GPCRK(GRK)是如何调节的 正常和病变的心肌信号和功能。这些激酶通过以下途径规范地调节GPCRs 磷酸化导致脱敏和反应丧失。GRK2和GRK5是主要的GRK 在心脏中表达，两者都被发现在衰竭的人类心肌中上调表达，也在 动物模型。最近，我们已经确定了GRK5在心脏中作为病理促进器的非GPCR作用。 肥大和向心衰的不良适应转变通过其在核内的定位和活动 心肌细胞。事实上，肥大应激诱导GRK5的核转位，其中GRK5可以作为一类 组蛋白脱乙酰酶(HDAC)激酶通过MEF2抑制肥大基因转录。 此外，GRK5以一种明显的非催化方式，可以促进肥厚时核NFAT的活性 基因转录。有趣的是，GRK5已经被证明直接与DNA结合，新的初步数据已经 通过染色质沉淀和测序(CHIP-SEQ)鉴定了几个基因靶点。病理性 在GRK5中，压力超载最小，似乎主要是通过其核局部化，这似乎 依赖于钙-钙调蛋白(CaM)与GRK5(GRK5nt)氨基末端区域的结合。 目前尚不清楚GRK5或其核活动是否参与了缺血损伤后的心脏病理 这将在这份提案中确定，我们有初步数据表明GRK5在 心肌细胞通过调节免疫细胞的侵袭和调节在缺血后心力衰竭中发挥新的作用 发炎。有趣的是，这种病理与GRK2显著不同。我们有现成的和新奇的 我们可以用小鼠模型来确定核GRK5在心力衰竭中的作用及其是否具有病理意义 机制涉及不需要GRK5催化活性的途径和基因靶点。这些型号 将在这个项目中被用来调查这些小说方面的GRK5在心力衰竭的发展后应激和 为了检验我们的中心假设，即GRK5在心脏的反应中发挥关键(病理)生理作用 损伤、靶向和操纵其在心肌细胞中的独特细胞定位和/或活性是一种新的 受伤心脏的治疗性修复策略。我们相关的具体目标是：[1]确定DNA/基因 肥大中GRK5的核靶点，并确定其基因调控中是否涉及激酶活性； [2]确定翻译方法是否可以用于治疗限制病理性核GRK5 [3]确定GRK5在心肌缺血损伤病理生理机制中的作用 以及这与GRK2介导的损伤有何具体不同。

Project 1 - SUMMARY The clinical syndrome heart failure (HF) continues to be a major health problem world-wide. It is an end-point after cardiac injury/stress, which is usually preceded by cardiac hypertrophy and adverse ventricular remodeling. Over the last two decades, our laboratory has studied HF development with a focus on the role of G protein- coupled receptor (GPCR) signaling. In particular, we are interested in how GPCR kinases (GRKs) regulate normal and diseased myocardial signaling and function. These kinases canonically regulate GPCRs via phosphorylation causing desensitization and a loss of response. GRK2 and GRK5 are the major GRKs expressed in the heart and both have been found to be up-regulated in failing human myocardium and also in animal models. Recently, we have identified a non-GPCR role for GRK5 in the heart as a pathological facilitator of hypertrophy and the maladaptive transition to HF via its localization and activity in the nucleus of cardiomyocytes. In fact, hypertrophic stress induces nuclear translocation of GRK5 where it can act as a Class II histone deacetylase (HDAC) kinase causing de-repression of hypertrophic gene transcription through MEF2. In addition, GRK5, in an apparent non-catalytic manner, can facilitate nuclear NFAT activity on hypertrophic gene transcription. Of interest, GRK5 has been shown to bind to DNA directly and new preliminary data has identified several gene targets via chromatin precipitation and sequencing (ChIP-Seq). The pathological nature of GRK5, a least in pressure-overload, appears to be primarily through its nuclear localization, which appears dependent on calcium-calmodulin (Ca2+-CaM) binding to a region within the amino-terminus of GRK5 (GRK5nt). What isn’t known is whether GRK5 or its nuclear activity participates in cardiac pathology after ischemic injury and this will be determined in this proposal where we have preliminary data suggesting increased GRK5 in myocytes plays a novel role in post-ischemic HF via regulation and modulation of immune cell infiltration and inflammation. Interestingly, this pathology differs significantly from that of GRK2. We have existing and novel mouse models available to us to determine the role of nuclear GRK5 in HF and whether its pathological mechanisms involve pathways and gene targets that do not require the catalytic activity of GRK5. These models will be employed in this Project to investigate these novels aspects of GRK5 in HF development post-stress and to test our Central Hypothesis that GRK5 plays a critical (patho)-physiological role in the heart’s response to injury and targeting and manipulating its unique cellular localization and/or activity in myocytes is a novel therapeutic repair strategy for the injured heart. Our associated Specific Aims are: [1] to determine the DNA/gene targets of nuclear GRK5 in hypertrophy and determine whether kinase activity is involved in its gene regulation; [2] To determine if a translational approach can be employed therapeutically to limit pathological nuclear GRK5 activity; [3] To determine the mechanistic action of GRK5 in the pathophysiology of myocardial ischemic injury and how this specifically differs from GRK2-mediated injury.