Role of the mAKAP Complex in Cardiac Hypertrophy

描述(申请人提供)：由A-激酶锚定蛋白(AKAP)形成的称为信号小体的多分子复合体有助于第二信使cAMP对细胞内信号的空间和时间限制。靶向存在于单个信号小体中的独特的蛋白质-蛋白质相互作用可能构成一种新的药物发现方法，产生一类新的选择性心脏疗法，显示出最小的非靶向副作用。其中一个信号体是由mAKAPb组成的，mAKAPb是一种结合腺酰环化酶5的支架蛋白，cAMP依赖的蛋白激酶A和Epac1，以及cAMP特异性的磷酸二酯酶PDE4D3。通过包括cAMP合成、降解和功能所必需的所有酶，mAKAPb复合体可以自主调节和响应局部控制的cAMP水平。MAKAPb信号体还含有ERK5丝裂原活化蛋白激酶和钙离子？依赖性磷酸酶钙调神经磷酸酶抗体。因此，肾上腺素能和gp130细胞因子/白血病抑制因子受体在体外诱导新生心肌细胞肥大的作用受到mAKAPb表达的RNAi的影响。这一应用有三个特定的目的，解决了两个中心假设：(1)mAKAPb在体内心脏重构的调节中发挥关键作用；(2)mAKAPb信号小体形成一个自主的cAMP信号室，其干扰将导致局部cAMP水平和整体心肌细胞表型的变化。具体目的1：mAKAPb在心脏重构中的作用。MAKAPb支架与病理重塑的体内相关性将在小鼠身上使用一种新的“牙线状”mAKAP等位基因进行测试。MAKAP基因将通过心脏特异的cre转基因被删除，并将对无应激、衰老小鼠和遭受慢性异丙肾上腺素输注、横动脉收缩和心肌梗死的小鼠进行研究。具体目标2：mAKAPb复合体对AC5的调节。AC5直接与mAKAPb中的N-末端结构域结合。AC5活性如何由结合的mAKAPb调节，将在体外和体内利用一种新型的转基因小鼠进行研究，在这种转基因小鼠中，AC5结合的多肽在心脏有条件地表达。具体目标3：mAKAPb信号体控制局部cAMP水平。MAKAPb信号体对活细胞内局部cAMP水平的调节将通过与mAKAPb融合的cAMP FRET传感器在培养的成人和新生心肌细胞中的表达来研究。使用野生型mAKAPb融合传感器获得的信号将与使用缺乏单个结合伙伴结合位点的mAKAPb传感器获得的信号进行比较，从而揭示单个支架蛋白复合体的破坏如何影响细胞内信号传递。通过这些特定目的获得的数据将建立mAKAPb信号小体作为肥大信号网络中的重要节点，并成为治疗适应性不良重塑和预防心力衰竭的特定药物治疗的候选靶点。 与公共卫生相关：心力衰竭是一种对公共健康具有重大意义的综合征，每年导致近30万人死亡。据估计，有570万美国公民患有心力衰竭，每年新增诊断病例近67万例。更好地了解控制心脏重构的细胞机制，包括心肌细胞肥大，可能会产生更好的治疗方案，降低死亡率。

DESCRIPTION (provided by applicant): The formation of multimolecular complexes called "signalosomes" by A-kinase anchoring proteins (AKAPs) contributes to the spatial and temporal restriction of intracellular signaling by the second messenger cAMP. Targeting unique protein-protein interactions present within individual signalosomes may constitute a novel approach to drug discovery, yielding a new class of selective cardiac therapies displaying minimal off- target side-effects. One such signalosome is organized by mAKAPb, a scaffold protein that binds adenylyl cyclase 5, the cAMP-dependent enzymes protein kinase A and Epac1, and the cAMP-specific phosphodiesterase PDE4D3. By including all of the enzymes necessary for cAMP synthesis, degradation, and function, mAKAPb complexes may autonomously regulate and respond to locally controlled cAMP levels. mAKAPb signalosomes also contain ERK5 mitogen-activated protein kinase and the Ca2???? dependent phosphatase calcineurin Ab. Accordingly, the induction of neonatal myocyte hypertrophy in vitro by adrenergic and gp130 cytokine/leukemia inhibitory factor receptors is impaired by RNAi of mAKAPb expression. This application has three Specific Aims that address two central hypotheses: (1) that mAKAPb plays a critical role in the regulation of cardiac remodeling in vivo, and (2) that the mAKAPb signalosome forms an autonomous cAMP signaling compartment whose disruption will result in changes both in local cAMP levels and overall myocyte phenotype. Specific Aim 1: The role of mAKAPb in cardiac remodeling. The in vivo relevance of the mAKAPb scaffold to pathologic remodeling will be tested in mice using a new "floxed" mAKAP allele. The mAKAP gene will be deleted using a cardiac-specific cre transgene, and both unstressed, aging mice and mice subjected to chronic isoproterenol infusion, transverse aortic constriction and myocardial infarction will be studied. Specific Aim 2: Regulation of AC5 by mAKAPb Complexes. AC5 directly binds to a N-terminal domain in mAKAPb. How AC5 activity is regulated by binding mAKAPb will be investigated in vitro and in vivo using a novel transgenic mouse in which an AC5-binding peptide is conditionally expressed in the heart. Specific Aim 3: Control of local cAMP levels by the mAKAPb signalosome. mAKAPb signalosome regulation of local cAMP levels in living cells will be investigated by the expression in cultured adult and neonatal cardiac myocytes of cAMP FRET sensors fused to mAKAPb. Signals obtained with a wildtype mAKAPb fusion sensor will be compared to that obtained using mAKAPb sensors lacking binding sites for individual binding partners, thereby revealing how the disruption of an individual scaffold protein complex affects intracellular signaling. Data obtained by these Specific Aims should establish the mAKAPb signalosome as an important node in the hypertrophic signaling network and as a candidate target for specific drug therapy for maladaptive remodeling and the prevention of heart failure. PUBLIC HEALTH RELEVANCE: Heart failure is a syndrome of major public heath significance accountable for nearly 300,000 deaths each year. It is estimated that 5.7 million US citizens suffer from heart failure, with nearly 670,000 new cases diagnosed annually. A better understanding of the cellular mechanisms that control cardiac remodeling, including myocyte hypertrophy, may yield better therapeutic regimens with decreased mortality.