Glycogen metabolism and PRKAG2 cardiomyopathy

说明（由申请人提供）：AMP活化蛋白激酶（AMPK）是一种丝氨酸/苏氨酸激酶，在包括心肌细胞在内的多种细胞类型中充当细胞能量传感器和代谢的主要调节剂。AMPK响应于细胞AMP/ATP比率的降低，这是能量状态受损的超敏感指标，并通过刺激ATP生成途径同时抑制ATP消耗途径来触发多个信号级联以恢复能量平衡。AMPK是由催化亚基（a）和两个调节亚基（P和v）组成的异源三聚体蛋白。每个亚基有2-3种亚型，除γ 3外，所有亚型都在心脏中表达。γ 2亚基（由PRKAG 2基因编码）的突变导致人类心肌病，其特征在于大量的心肌糖原积累、预激综合征和心脏肥大。几个实验室的研究表明，人类疾病的表型可以忠实地再现在转基因小鼠与心脏特异性过表达的突变型γ 2亚基的AMPK。使用在心脏中表达N488 I突变体v2-AMPK的小鼠模型，我们已经表明，在没有能量缺陷的情况下，突变导致AMPK的异常激活，并且可以通过引入AMPK的失活催化亚基来挽救疾病表型。然而，AMPK活性改变导致心肌病的机制仍然未知。虽然AMPK在能量应激过程中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确立，但对能量完整的心脏中AMPK活性异常的代谢后果知之甚少。在这里，我们提出，由于PRKAG 2突变异常AMPK活性重塑心脏代谢，以实现一个新的水平的能量稳态，建立和打开一个大大扩大的糖原池的代价发展糖原累积型心肌病。为了验证这一假设，我们将结合联合收割机假设驱动和基于发现的方法来剖析导致表达PRKAG 2的N488 I突变体的小鼠心脏中糖原储存的代谢途径。我们将首先使用31 P和13 C NMR光谱结合生物化学方法表征突变小鼠心脏的重塑代谢谱。其次，我们将确定候选分子介导的糖原积累，通过鉴定靶蛋白和转录的变化，可能直接或间接导致异常AMPK活性。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物化学和分子生物学分析，评估候选蛋白在细胞代谢中的作用。从这项研究中获得的结果将有助于我们了解生理学和疾病中心脏特异性AMPK信号传导的长期目标。

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.