Inhibition of GRK2 to Prevent Ventricular Remodeling and Heart Failure After CABG

描述（由申请人提供）：尽管通过冠状动脉旁路移植术（CABG）实现了完全血运重建，但心肌梗死后左心室功能下降的患者的长期生存率明显较低。不良或适应不良的心室重构可在血运重建后持续，似乎是CABG后进展为HF的长期结局不良的重要风险因素。心脏成纤维细胞（CF）占心脏总细胞量的60-70%，在调节正常心肌功能和心肌梗死发生的不良重塑以及向HF转变中发挥关键作用。CF导致的过度胶原沉积导致心肌硬化、舒张功能障碍和心脏超负荷，这可能是由于负责基础细胞外基质（ECM）稳态的静止成纤维细胞转化为活化的肌成纤维细胞（myoFb）的结果。在组织如心脏中需要抑制这种转化的方法，其中CF产生的过量ECM导致纤维化和重塑，这是HF的前兆。最近的研究表明，细胞内cAMP的增加和下游cAMP依赖性蛋白激酶（PKA）的激活可以抑制体外myoFb的形成和胶原蛋白的合成。CF中cAMP产生的主要机制是通过刺激与腺苷酸环化酶偶联的膜结合22-肾上腺素能受体。我们实验室的最新数据显示，2-肾上腺素能受体（2-AR）信号传导和cAMP产生在从晚期HF患者心室分离的CF中严重受损，可能是myoFb形成和ECM沉积增加的关键潜在机制。我们已经发现，G蛋白偶联受体激酶-2（GRK 2），一种丝氨酸-苏氨酸激酶，磷酸化和解偶联激动剂占据的2-AR，在CF中强烈表达，GRK 2活性在慢性HF中显著上调。该项目的长期目标是明确描述2-AR信号传导和GRK 2在成人CF转化为myoFb中的作用，目的是抑制或最小化病理性纤维化，尽管成功血运重建，但病理性纤维化可能导致心肌梗死后HF。我们的中心假设是，受损的2-AR信号，作为GRK 2活性增加的结果，介导CF到myoFb的转化，并增加ECM的合成。因此，受损的CF2-AR信号传导促进适应不良的重构和进行性心功能障碍，从而导致HF.这将使用分子方法来测试，以在从正常和衰竭的人心室分离的CF中敲低和过表达GRK 2，以研究GRK 2在调节CF激活中的作用。将在大鼠心肌梗死模型中进行体内研究，使用腺相关病毒以CF特异性方式转移GRK 2的微型基因抑制剂，以实现长期表达并评估抑制不良重塑和改善心脏功能和存活率的潜在治疗功效。抑制GRK 2活性在这种疾病过程中具有重要的治疗前景，并可能代表CABG时的新的预防性治疗。 公共卫生相关性：这项拟议的研究与公共卫生有关，因为发现了一种新的预防性治疗方法，可以在冠状动脉搭桥手术时提供，以抑制患有术前心肌梗死的患者向心力衰竭的潜在转变，可以显著提高生活质量和长期生存率。这项研究也可能与预防其他器官的纤维化有关，这些器官也会导致显著的发病率和死亡率。

DESCRIPTION (provided by applicant): Despite complete revascularization by coronary artery bypass grafting (CABG), long-term survival is markedly inferior for patients who present with depressed left ventricular function post-myocardial infarction. Adverse or maladaptive ventricular remodeling can continue following revascularization and appears to be an important risk factor for poor long-term outcomes after CABG with progression to HF. Cardiac fibroblasts (CF) make up 60-70% of the total cell mass of the heart and play a critical role in regulating normal myocardial function and in the adverse remodeling that occurs with myocardial infarction and the transition to HF. Excessive collagen deposition by CF leads to myocardial stiffening, diastolic dysfunction, and overload of the heart, likely as a consequence of transformation of quiescent fibroblasts responsible for basal extracellular matrix (ECM) homeostasis to activated myofibroblasts (myoFb). Approaches to inhibit this transformation are needed in tissues, such as the heart, where excessive ECM production by CF leads to fibrosis and remodeling which is a precursor to HF. Recent work has demonstrated that increased intracellular cAMP and downstream activation of cAMP-dependent protein kinase (PKA) can inhibit myoFb formation and collagen synthesis in vitro. The primary mechanism for cAMP production in CF is through stimulation of membrane-bound 22-adrenergic receptors which couple to adenylyl cyclase. Recent data from our laboratory show that 2-adrenergic receptor (2-AR) signaling and cAMP production are severely impaired in CF isolated from ventricles of patients with advanced HF and may be a critical underlying mechanism for myoFb formation and increased ECM deposition. We have found that G protein-coupled receptor kinase-2 (GRK2), a serine-threonine kinase that phosphorylates and uncouples agonist-occupied 2-ARs, is robustly expressed in CF and GRK2 activity is significantly upregulated in chronic HF. The long-term goal of this project is to specifically delineate the roles of 2-AR signaling and GRK2 in adult CF transformation to myoFb with the aim of inhibiting or minimizing pathological fibrosis that can lead to HF post-myocardial infarction despite successful revascularization. Our central hypothesis is that impaired 2-AR signaling, as a result of increased GRK2 activity, mediates CF to myoFb transformation and increases ECM synthesis. Thus, impaired CF 2-AR signaling promotes maladaptive remodeling and progressive cardiac dysfunction contributing to HF. This will be tested using molecular approaches to knockdown and over express GRK2 in CF isolated from normal and failing human ventricles to investigate the role of GRK2 in regulating CF activation. In vivo studies will be performed in a rat myocardial infarction model using adeno-associated viral gene transfer of a mini-gene inhibitor of GRK2 in a CF-specific manner to achieve long-term expression and evaluate potential therapeutic efficacy in inhibiting adverse remodeling and improving cardiac function and survival. Inhibition of GRK2 activity has important therapeutic promise in this disease process and may represent a novel adjunctive therapy at the time of CABG. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the discovery of a novel adjunctive therapy which could be delivered at the time of coronary artery bypass surgery to inhibit the potential transition to heart failure in patients who have suffered a pre-operative myocardial infarction could lead to significantly greater quality of life and long-term survival. This research may also be relevant to preventing fibrosis of other organs which also contribute to significant morbidity and mortality.