Isoform-specific function of y-AMPK in the heart.

说明(申请人提供)：AMPK是一种丝氨酸/苏氨酸激酶，作为细胞内能量感受器和细胞新陈代谢、生长和死亡的关键调节因子，因此是治疗肥胖、糖尿病、癌症和心血管疾病的有前景的药物靶点。AMPK是由催化的1亚基和调节的2和3亚基组成的异三聚体复合体，每个亚基有多种异构体。以往的研究表明，应激时心脏中AMPK的激活是一种重要的心脏保护机制。然而，AMPK调节性32亚基(由prkag2基因编码)的点突变会导致以心肌肥大、心律失常和糖原储存为特征的人类心肌病。这引起了人们对未来针对心脏AMPK级联反应的药物开发的担忧。因此，了解prkag2心肌病的发病机制至关重要。利用在心脏过度表达突变体prkag2(N488I)的转基因小鼠(TG32N488I)忠实地概括了prkag2心肌病，我们在之前的资助期间证明了该突变导致AMPK异常激活，在没有能量缺乏的情况下，通过抑制AMPK活性可以挽救心肌病的表型。我们进一步证明，在没有能量应激的情况下，葡萄糖摄取和脂肪酸氧化的同时增加导致了TG32N488I心脏中外源葡萄糖优先进入糖原池的代谢重路由。为了确定心肌病表型是否完全归因于糖原储存，我们试图通过特异性靶向肌肉糖原合成酶的活性来在不改变突变基因表达的情况下挽救糖原储存。我们的初步数据显示，该策略成功地使TG32N488I小鼠的心肌糖原含量正常化，但不能使心肌肥大表型正常化。因此，我们假设32-AMPK突变引起的心肌肥厚与糖原储存无关。此外，由于prkag2突变主要引起心脏表型，它提出了32-AMPK在心脏中的独特作用的问题。这导致我们建立了32-AMPK缺乏的小鼠模型，以确定3-AMPK的异构体特异性功能。利用这些模型，我们提出了1)检验prkag2突变导致心肌肥大不依赖于糖原储存的假设，并确定在营养饱和条件下32-AMPK异常激活刺激心肌肥大的机制；2)确定心脏发育和疾病期间心脏中32-AMPK信号级联的组成和功能。 公共卫生相关性：该项目调查由编码AMP激活的蛋白激酶(AMPK)的G2亚单位基因突变引起的人类心肌病的发病机制。由于AMPK是治疗代谢紊乱、心血管疾病和癌症的有前景的药物靶点，本项目还将确定G2-AMPK信号通路在心脏中的信号机制，为新的治疗策略提供基础。

DESCRIPTION (provided by applicant): AMP-activated protein kinase (AMPK) is serine/threonine kinase that serves as an intracellular energy sensor and a key regulator of cell metabolism, growth and death, hence a promising drug target for obesity, diabetes, cancer and cardiovascular diseases. AMPK is a heterotrimeric complex composed of catalytic 1-subunit and regulatory 2- and 3-subunits with multiple isoforms for each subunit. Previous studies have shown that activation of AMPK in the heart during stress is an important cardioprotective mechanism. However, point mutations in the regulatory 32-subunit (encoded by prkag2 gene) of AMPK cause human cardiomyopathy characterized by cardiac hypertrophy, arrhythmias and glycogen storage. This raises concerns of future drug development targeting AMPK cascade in the heart. It is thus critical to understand the disease mechanisms responsible for the prkag2 cardiomyopathy. Using transgenic mice overexpressing mutant prkag2 (N488I) in the heart (TG32N488I) that faithfully recapitulated prkag2 cardiomyopathy, we in the previous funding period demonstrated that the mutation caused aberrant activation of AMPK in the absence of energetic deficit, and the cardiomyopathy phenotype could be rescued by inhibition of AMPK activity. We have further demonstrated that the simultaneous increases in glucose uptake and fatty acid oxidation in the absence of energy stress resulted in metabolic re-routing of exogenous glucose preferentially into glycogen pool in the TG32N488I hearts. To determine whether the cardiomyopathy phenotype is entirely attributable to glycogen storage, we sought to rescue the glycogen storage without changing the mutant gene expression by specifically targeting the activity of muscle glycogen synthase. Our preliminary data showed that this strategy successfully normalized cardiac glycogen content but not the cardiac hypertrophy phenotype in TG32N488I mice. Thus, we hypothesize that 32- AMPK mutation causes cardiac hypertrophy independent of glycogen storage. Moreover, since the prkag2 mutation causes predominantly cardiac phenotype, it raises the question of a unique role of 32-AMPK in the heart. This led us to generate mouse models deficient of 32-AMPK in order to determine the isoform-specific function of 3-AMPK. Using these models we propose 1) to test the hypothesis that the prkag2 mutation causes cardiac hypertrophy independent of glycogen storage and, to determine the mechanisms by which aberrant activation of 32-AMPK under nutrient saturated conditions stimulate cardiac hypertrophy; 2) to define the composition and the function of the 32-AMPK signaling cascade in the heart during cardiac development and diseases. PUBLIC HEALTH RELEVANCE: This project investigates disease mechanisms of human cardiomyopathy caused by mutations of the gene encoding g2-subunit of the AMP-activated protein kinase (AMPK). Because AMPK is a promising drug target for metabolic disorders, cardiovascular diseases and cancer, this project will also define the signaling mechanisms of g2-AMPK pathway in the heart in order to provide a basis for novel therapeutic strategies.