Role of the mAKAP Complex in Cardiac Hypertrophy

描述（由申请人提供）：A-激酶锚定蛋白（AKAP）形成称为“信号体”的多分子复合物，有助于第二信使cAMP对细胞内信号传导的空间和时间限制。靶向个体信号体内存在的独特蛋白质-蛋白质相互作用可能构成药物发现的新方法，产生一类显示出最小脱靶副作用的新的选择性心脏疗法。一种这样的信号体由mAKAPb组织，mAKAPb是结合腺苷酸环化酶5、cAMP依赖性酶蛋白激酶A和Epac 1以及cAMP特异性磷酸二酯酶PDE 4D 3的支架蛋白。通过包含cAMP合成、降解和功能所必需的所有酶，mAKAPb复合物可以自主调节和响应局部控制的cAMP水平。mAKAPb信号体还含有ERK 5丝裂原活化蛋白激酶和Ca 2+信号体。依赖性磷酸酶钙调磷酸酶抗体因此，通过mAKAPb表达的RNAi，在体外通过肾上腺素能和gp 130细胞因子/白血病抑制因子受体诱导新生心肌细胞肥大受到损害。本申请具有三个特定目的，其解决了两个中心假设：（1）mAKAPb在体内心脏重塑的调节中起关键作用，以及（2）mAKAPb信号体形成自主cAMP信号传导区室，其破坏将导致局部cAMP水平和总体肌细胞表型的变化。具体目标1：mAKAPb在心脏重塑中的作用。将使用新的“floxed”mAKAP等位基因在小鼠中测试mAKAPb支架与病理性重塑的体内相关性。将使用心脏特异性cre转基因删除mAKAP基因，并且将研究未应激的衰老小鼠和经受慢性异丙肾上腺素输注、横向主动脉缩窄和心肌梗死的小鼠。具体目标2：通过mAKAPb复合物调节AC 5。AC 5直接结合mAKAPb中的N-末端结构域。如何AC 5活性的调节结合mAKAPb将在体外和体内研究使用一种新的转基因小鼠，其中AC 5结合肽有条件地表达在心脏。具体目标3：通过mAKAPb信号体控制局部cAMP水平。将通过在培养的成人和新生儿心肌细胞中表达与mAKAPb融合的cAMP FRET传感器来研究活细胞中局部cAMP水平的mAKAPb信号体调节。将用野生型mAKAPb融合传感器获得的信号与使用缺乏单个结合配偶体的结合位点的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物的破坏如何影响细胞内信号传导。通过这些特定目的获得的数据应将mAKAPb信号体确立为肥大信号网络中的重要节点，并作为适应不良重塑和预防心力衰竭的特异性药物治疗的候选靶点。 公共卫生相关性：心力衰竭是一种具有重大公共卫生意义的综合征，每年造成近30万人死亡。据估计，570万美国公民患有心力衰竭，每年诊断出近67万例新病例。更好地理解控制心脏重塑的细胞机制，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.