Regulation of Beta Adrenergic Receptor Function by localized P13K Inhibition

通过局部 P13K 抑制调节 β 肾上腺素能受体功能

• 批准号: 7492819
• 负责人: Howard A Rockman
• 金额: $ 40.83万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7492819
• 关键词: 1-Phosphatidylinositol 4-Kinase; Adenylate Cyclase; Agonist; Animal Model; Binding; Calcineurin; Calcineurin inhibitor; Calcium; Calsequestrin; Cardiac; Cardiac Myocytes; Catecholamines; Cell Nucleus; Cells; Chronic; Clathrin; Complex; Constriction procedure; Correlative Study; Coupling; Data; Dominant-Negative Mutation; Down-Regulation; Endocytosis; Evaluation; Event; Exercise; Failure; Family suidae; GTP-Binding Proteins; Gene Expression; Gene Transfer; Generations; Heart; Heart Hypertrophy; Heart failure; Human; Hypertrophy; In Vitro; Indium; Infarction; Knock-out; Knowledge; Lead; Lipids; Localized; Mediating; Modeling; Molecular; Mus; Muscle Cells; NF-AT; Numbers; Oryctolagus cuniculus; Pathogenesis; Peptides; Performance; Phenotype; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Physiological; Pik-off; Play; Process; Protein Kinase; Protein Overexpression; Proteins; Protocols documentation; Regulation; Rodent Model; Role; Signal Pathway; Signal Transduction; Stimulus; Sus scrofa; TFAP2A gene; TRP channel; TRPC3 ion channel; Tertiary Protein Structure; Testing; Transactivation; Transgenes; Transgenic Animals; Transgenic Mice; adenoviral-mediated; beta-adrenergic receptor; beta-arrestin; coated pit; desensitization; gain of function; in vivo; loss of function; mouse model; muscle LIM protein; mutant; nuclear factors of activated T-cells; pressure; prevent; promoter; receptor; receptor function; receptor internalization; response; transcription factor

Beta-adrenergic receptor (betaAR) signaling plays an important role in the regulation of cardiac performance. BetaAR downregulation and desensitization resulting in reduced responsiveness to catecholamines are hallmarks of the failing heart. In human heart failure, diminished receptor number (downregulation) and impaired coupling (desensitization) of betaARs result in reduced responsiveness to catecholamines. The process that initiates desensitization of the receptor begins with the phosphorylation of betaARs by beta-adrenergic receptor kinase1 (betaARK1), followed by the binding of beta-arrestin, which together inhibits coupling to G protein and loss of effector (adenylyl cyclase) signaling. The beta-arrestin receptor complex is then targeted to the clathrin-coated pits for endocytosis through a process involving interaction of beta-arrestin with AP-2 and clathrin. Recently we have shown that betaARK1 interacts with the lipid kinase, phosphoinositide 3-kinase (PI3K), to form a cytosolic complex. We have shown that the betaARK/PI3K interaction is critical for the agonist-mediated internalization of betaARs and if the betaARK/PI3K interaction is disrupted in vivo, betaARs do not undergo downregulation and desensitization with chronic catecholamine stimulation. Importantly, PI3K contains both lipid kinase and protein kinase activity. Although we have shown that the lipid products of PI3K are important for receptor internalization, it is not yet known whether the protein kinase activity of PI3K is necessary for betaAR endocytosis and activation of downstream signaling pathways. Since receptor-localized PI3K plays a critical role in betaAR internalization, knowledge of the distinct roles of the protein and lipid kinase activities in the process of betaAR sequestration, resensitization and signaling would contribute to a better understanding of the regulation of betaAR function in the pathogenesis of heart failure. Our Central Hypothesis is that downregulation of cardiac betaARs, in vivo, can be prevented through inhibition of the local generation of D-3 phosphatidylinositols by PI3K within the receptor complex and preserving betaAR function in the failing heart will lead to an amelioration of heart failure in both small and large animal models of cardiac failure. We will test the hypothesis that the protein kinase activity of PI3K is required for betaAR internalization and downregulation, and that betaAR function will be normal in hearts displaying a "physiological" hypertrophic phenotype compared to hearts that show a "pathological" hypertrophic phenotype. Specific Aims (1) To determine the molecular mechanisms of PI3K-mediated betaAR internalization by studying PI3K mutants that lack either protein kinase activity or lipid kinase activity; (2) To determine whether overexpression of the minimal peptide of PI3K (PIK domain peptide, PIKdp), which acts as a dominant negative in vitro, will prevent betaAR downregulation in vivo and rescue mouse models of heart failure: a) chronic pressure overload (transverse aortic constriction, TAC), b) Calsequestrin overexpression X PIKdp, c) muscle LIM protein knock out X PIKdp; (3) To determine whether overexpression of PI3Kdp in vivo by adenoviral-mediated gene transfer to the heart will prevent betaAR downregulation and rescue two large animal models of cardiac failure: a) AdenoPIKdp injected into a rabbit infarct model, b) the pacing pig heart failure model; (4) To determine whether the mechanism that causes transition from hypertrophy to heart failure is due to the type of load on the heart (exercise vs. pressure) or the chronicity of the load. Chronic TAC will be compared to intermittent TAC and two exercise protocols in mice, together with a comprehensive evaluation of the developing cardiac phenotype.

β-肾上腺素能受体（BETAAR）信号传导在心脏性能的调节中起重要作用。贝塔尔下调和脱敏导致对儿茶酚胺的反应性降低是失败的心脏的标志。在人类心力衰竭中，受体数量（下调）和βAR的偶联（脱敏）导致对儿茶酚胺的反应性降低。启动受体脱敏的过程始于β-肾上腺素能受体激酶1（Betaark1）的βAR磷酸化，然后是β-arrestin的结合，共同抑制了与G蛋白和效应损失（adenylylylyl Cyclase）信号的偶联。然后，通过涉及β-arrestin与AP-2和Clathrin的相互作用的过程，将β-arrestin受体复合物靶向涂有网格蛋白涂层的凹坑以进行内吞作用。最近，我们表明betaark1与脂质激酶，磷酸肌醇3-激酶（PI3K）相互作用，形成了胞质复合物。我们已经表明，Betaark/PI3K相互作用对于激动剂介导的Betaars的内在化至关重要 Betaark/PI3K相互作用在体内破坏，βAR不会通过慢性儿茶酚胺刺激进行下调和脱敏。重要的是，PI3K既包含脂质激酶和蛋白激酶活性。尽管我们已经表明，PI3K的脂质产物对于受体内在化很重要，但尚不清楚PI3K的蛋白激酶活性是否对于βAR内吞作用和下游信号传导途径的激活是必需的。由于受体定位的PI3K在betaar内部化中起着至关重要的作用，因此了解 蛋白质和脂质激酶活性在βAR隔离过程，敏化和信号传导过程中的不同作用将有助于更好地理解βAR功能在心力衰竭发病机理中的调节。我们的中心假设是，可以通过抑制局部产生的受体复合物中PI3K的局部生成D-3磷脂酰肌醇的局部，并保留βAR在失败的心脏中的功能将导致小型和大型心脏动物模型的心脏失效。我们将检验以下假设：BetaAR内在化和下调所必需的PI3K的蛋白激酶活性，并且与表现出“病理学”肥大性表型相比的心脏中，Betaar功能将在心脏中正常，表现出“生理”肥大表型。通过研究缺乏蛋白激酶活性或脂质激酶活性的PI3K突变体来确定PI3K介导的βAR内在化的分子机制的具体目的（1）； （2）确定PI3K最小肽的过表达（PIK结构域肽，PIKDP）是否充当主要阴性 在体外，将防止体内的βAR下调和拯救心力衰竭的小鼠模型：a）慢性压力超负荷（横向主动脉收缩，TAC），b）cal sequesterin Extrentin过表达x pikdp，c）肌肉lim蛋白敲除x pikdp； （3）确定通过腺病毒介导的基因转移到心脏中PI3KDP在体内的过表达是否会防止βAR的下调并挽救两种大动物心脏衰竭的大动物模型：a）将adenopikdP注入兔子梗塞模型中，b）起搏猪心力衰竭模型； （4）确定导致从肥大到心力衰竭过渡的机制是由于心脏上的负载类型（锻炼与压力）或负载的慢性造成的。将慢性TAC与小鼠的间歇性TAC和两种运动方案进行比较，并全面评估发育中的心脏表型。