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）进行完全血运重建，但心肌梗死后出现左心室功能下降的患者的长期生存率明显较低。不良或不适应的心室重构可在血运重建术后继续存在，这似乎是冠状动脉搭桥术进展为心衰后不良长期预后的重要危险因素。心脏成纤维细胞（CF）占心脏总细胞质量的60-70%，在调节正常心肌功能以及心肌梗死和向心衰过渡时发生的不良重构中发挥关键作用。CF导致过多的胶原沉积导致心肌硬化、舒张功能障碍和心脏负荷过重，这可能是负责基底细胞外基质（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。在组织中需要抑制这种转化的方法，如心脏，CF产生过多的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cAMP依赖性蛋白激酶（PKA）的下游活化可以在体外抑制myoFb的形成和胶原合成。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。我们实验室最近的数据显示，从晚期HF患者心室分离的CF中，2-肾上腺素能受体（2-AR）信号和cAMP的产生严重受损，这可能是myoFb形成和ECM沉积增加的关键潜在机制。我们发现G蛋白偶联受体激酶-2 （GRK2）是一种丝氨酸-苏氨酸激酶，可磷酸化和解偶联激动剂占据的2-ARs，在CF中强烈表达，而GRK2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信号和GRK2在成人CF向myoFb转化中的作用，目的是抑制或减少病理性纤维化，病理性纤维化可导致心肌梗死后的HF，尽管成功的血运重建。我们的中心假设是，由于GRK2活性增加，2-AR信号受损，介导CF到myoFb的转化并增加ECM合成。因此，受损的CF 2-AR信号可促进不适应重构和进行性心功能障碍，从而导致HF。这将通过分子方法进行测试，在从正常和衰竭的人心室分离的CF中敲除和过表达GRK2，以研究GRK2在调节CF激活中的作用。体内研究将在大鼠心肌梗死模型中进行，使用腺相关病毒以cf特异性方式转移GRK2的迷你基因抑制剂，以实现长期表达，并评估抑制不良重构和改善心功能和生存的潜在治疗效果。抑制GRK2活性在这种疾病过程中具有重要的治疗前景，可能是一种新的辅助治疗方法。

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.