Role of the mAKAP Complex in Cardiac Hypertrophy

mAKAP 复合物在心脏肥大中的作用

• 批准号: 8299972
• 负责人: Michael Seth Kapiloff
• 金额: $ 37.87万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-08 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299972
• 关键词: A kinase anchoring protein; Acute; Address; Adenylate Cyclase; Adrenergic Agents; Adrenergic Receptor; Adult; Adverse effects; Affect; Aging; Agonist; Alleles; Apoptosis; Attenuated; Binding; Binding Sites; Calcineurin; Calmodulin; Cardiac; Cardiac Myocytes; Cells; Cessation of life; Chronic; Complex; Critical Care; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Diagnosis; Dobutamine; Dopamine; Drug usage; Echocardiography; Elements; Enzymes; Feedback; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Guanine Nucleotide Exchange Factors; Heart; Heart Hypertrophy; Heart failure; Histopathology; Hypertrophy; In Vitro; Individual; Infusion procedures; Isoproterenol; Laboratories; Life; MAPK7 gene; Maps; Mitogen-Activated Protein Kinases; Mus; Muscle; Muscle Cells; Muscle function; Myocardial Infarction; Myocardium; N-terminal; Neonatal; Nuclear; PDE4D3; Pathologic; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Physiological; Play; Prevention; Protein Binding; RNA Interference; Regimen; Regulation; Role; Scaffolding Protein; Second Messenger Systems; Signal Transduction; Site; Specificity; Stress; Syndrome; Testing; Therapeutic; Time; Transgenes; Transgenic Mice; adenylyl cyclase type V; adrenergic; calcineurin phosphatase; constriction; cytokine; drug discovery; hemodynamics; in vivo; leukemia inhibitory factor receptor; mortality; novel; novel strategies; overexpression; phosphoric diester hydrolase; prevent; protein complex; protein protein interaction; public health relevance; scaffold; second messenger; sensor

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.

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。针对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，显示出最小的脱靶副作用。其中一个信号体由 mAKAPb 组织，mAKAPb 是一种结合腺苷酸环化酶 5、cAMP 依赖性酶蛋白激酶 A 和 Epac1 以及 cAMP 特异性磷酸二酯酶 PDE4D3 的支架蛋白。通过包含 cAMP 合成、降解和功能所需的所有酶，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 ERK5 丝裂原激活蛋白激酶和 Ca2？？？依赖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儿肌细胞肥大会受到 mAKAPb 表达的 RNAi 的损害。该申请有三个具体目标，解决了两个中心假设：(1) mAKAPb 在体内心脏重塑的调节中发挥着关键作用，(2) mAKAPb 信号体形成自主的 cAMP 信号室，其破坏将导致局部 cAMP 水平和整体心肌细胞表型的变化。具体目标 1：mAKAPb 在心脏重塑中的作用。 mAKAPb 支架与病理重塑的体内相关性将在小鼠中使用新的“floxed”mAKAP 等位基因进行测试。将使用心脏特异性的 cre 转基因删除 mAKAP 基因，并对无应激的衰老小鼠和接受长期异丙肾上腺素输注、横向主动脉缩窄和心肌梗塞的小鼠进行研究。具体目标 2：mAKAPb 复合物对 AC5 的调节。 AC5 直接结合 mAKAPb 中的 N 端结构域。将使用新型转基因小鼠在体外和体内研究如何通过结合 mAKAPb 来调节 AC5 活性，其中 AC5 结合肽在心脏中条件性表达。具体目标 3：mAKAPb 信号体控制局部 cAMP 水平。将通过与mAKAPb融合的cAMP FRET传感器在培养的成人和新生儿心肌细胞中的表达来研究mAKAPb信号体对活细胞中局部cAMP水平的调节。将使用野生型 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。通过这些特定目标获得的数据应将 mAKAPb 信号体确立为肥厚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定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，每年导致近 30 万人死亡。据估计，美国有 570 万公民患有心力衰竭，每年新诊断病例近 67 万例。更好地了解控制心脏重塑（包括心肌细胞肥大）的细胞机制可能会产生更好的治疗方案并降低死亡率。