Glycogen metabolism and PRKAG2 cardiomyopathy

说明(申请人提供)：AMPK是一种丝氨酸/苏氨酸激酶，在包括心肌细胞在内的各种细胞类型中充当细胞能量感受器和新陈代谢的主调节器。AMPK对细胞内AMP/ATP比值的降低做出反应，AMP/ATP比值是能量受损的超灵敏指标，AMPK通过刺激ATP生成途径，抑制ATP消耗途径，触发多个信号级联反应，恢复能量平衡。AMPK是由一个催化亚基(A)和两个调节亚基(p和v)组成的异三聚体蛋白。每个亚基有2-3个亚型，除Y3外，其他亚基均在心脏表达。Y2亚基突变(由PRKAG2基因编码)会导致人类心肌病，其特征是大量心肌糖原积聚、预激综合征和心肌肥大。几个实验室的研究表明，AMPK突变的y2亚单位过表达的转基因小鼠可以真实地再现人类疾病的表型。通过在心脏中表达N488I突变体v2-AMPK的小鼠模型，我们发现在没有能量缺乏的情况下，该突变导致AMPK的异常激活，并且通过引入一个失活的AMPK催化亚基可以挽救疾病的表型。然而，AMPK活性改变导致心肌病的机制仍不清楚。虽然AMPK在能量应激期间促进葡萄糖和脂肪酸利用的重要作用在心肌和骨骼肌中得到了很好的证实，但对能量完整心脏中AMPK活性异常的代谢后果却知之甚少。在这里，我们认为PRKAG2突变引起的AMPK活性异常重塑心脏新陈代谢，通过建立和翻转显著扩大的糖原池来实现新的能量平衡水平，代价是发生糖原储存性心肌病。为了验证这一假设，我们将结合假设驱动和基于发现的方法来剖析表达PRKAG2的N488I突变的小鼠心脏中导致糖原储存的代谢途径。我们将首先使用31P和13C核磁共振波谱以及生化方法来表征突变小鼠心脏中重塑的代谢谱。其次，我们将通过识别目标蛋白和可能直接或间接导致AMPK活性异常的转录变化来定义介导糖原积累的候选分子。最后，我们将通过对候选蛋白在心肌细胞和小鼠心脏中的功能进行生化和分子生物学分析，来评估它们在细胞代谢中的作用。这项研究的结果将有助于我们理解心脏特异的AMPK信号在生理学和疾病中的长期目标。

DESCRIPTION (provided by applicant): AMP-activated protein kinase (AMPK) is a serine/threonine kinase that acts as a cellular energy sensor and a master regulator of metabolism in a variety of cell types including cardiac myocytes. AMPK responds to decreases in cellular AMP/ATP ratio, an ultra-sensitive indicator of impaired energy status, and triggers multiple signaling cascades to restore energy balance by stimulating ATP-generating pathways while inhibiting ATP consuming pathways. AMPK is a heterotrimeric protein consisting of a catalytic subunit (a) and two regulatory subunits (P and v). Each subunit has 2-3 isoforms, all except y3 are expressed in the heart. Mutations in the y2 subunit (encoded by PRKAG2 gene) cause human cardiomyopathy characterized by substantial myocardial glycogen accumulation, pre-excitation syndrome, and cardiac hypertrophy. Studies by several laboratories have shown that the human disease phenotype can be faithfully recapitulated in transgenic mice with cardiac-specific overexpression of the mutant y2 subunit of AMPK. Using the mouse model expressing N488I mutant v2-AMPK in the hearts, we have shown that the mutation causes aberrant activation of AMPK in the absence of energetic deficit, and the disease phenotype can be rescued by introducing an inactive catalytic subunit of AMPK. However, the mechanisms by which altered AMPK activity cause cardiomyopathy remains unknown. While the important role of AMPK in promoting glucose and fatty acids utilization during energetic stress is well established in cardiac and skeletal muscle, the metabolic consequence of inordinate AMPK activity in energetically intact heart is poorly understood. Here we propose that abnormal AMPK activity due to PRKAG2 mutation remodels cardiac metabolism to achieve a new level of energy homeostasis by establishing and turning over a substantially enlarged glycogen pool at the expense of developing glycogen storage cardiomyopathy. To test this hypothesis, we will combine hypothesis-driven and discovery-based approaches to dissect the metabolic pathways leading to glycogen storage in mouse hearts expressing N488I mutant of PRKAG2. We will first characterize the remodeled metabolic profile in mutant mouse hearts using 31P and 13C NMR spectroscopy together with biochemical approaches. Secondly, we will define candidate molecules that mediate glycogen accumulation by identifying target proteins and transcriptional changes that may result directly or indirectly from aberrant AMPK activity. Finally, we will evaluate the role of candidate proteins in cellular metabolism by biochemical and molecular biology analyses of their function in cardiac myocytes and mouse hearts. Results obtained from this study will serve our long-term goal of understanding the cardiac-specific AMPK signaling in physiology and disease.