Extracellular ubiquitin: role in myocyte apoptosis and myocardial remodeling

细胞外泛素：在心肌细胞凋亡和心肌重塑中的作用

• 批准号: 7589134
• 负责人: KRISHNA SINGH
• 金额: $ 21.3万
• 依托单位: EAST TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-10 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7589134
• 关键词: 1-Phosphatidylinositol 3-Kinase; Address; Adrenergic Receptor; Adult; Agonist; Angiotensin II; Apoptosis; Apoptotic; Area; Biochemical; Cardiac Myocytes; Cells; Cessation of life; Chronic; Conditioned Culture Media; Cytochromes; Data; Fibrosis; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Heart failure; Histology; Hydrogen Peroxide; In Vitro; Infusion procedures; Isoproterenol; JUN gene; Left Ventricular Dysfunction; Left Ventricular Function; Measures; Mediating; Mitochondria; Monoubiquitination; Morphology; Mus; Muscle Cells; Myocardial; Myocardium; Oxidative Stress; Pathway interactions; Patients; Play; Polyubiquitination; Proteins; Proteomics; Rattus; Role; Serum; Signal Pathway; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stimulus; Testing; Therapeutic; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Two-Dimensional Gel Electrophoresis; Ubiquitin; Ventricular; Ventricular Remodeling; Western Blotting; caspase-3; extracellular; in vivo; inhibitor/antagonist; insight; neutralizing antibody; novel; novel strategies; protein degradation; public health relevance; stress-activated protein kinase 1

DESCRIPTION (provided by applicant): Increased sympathetic activity is a central feature in patients with heart failure. Stimulation of ? adrenergic receptor (?-AR), specifically ?1-AR, increases cardiac myocyte apoptosis in vitro and in vivo, while stimulation of ?2-AR plays an anti-apoptotic role in ?-AR-stimulated apoptosis. We have shown that ?-AR-stimulated apoptosis is mediated via the activation of glycogen synthase kinase-3? (GSK-3?) and JNK-dependent mitochondrial death pathway in adult rat ventricular myocytes (ARVMs). ?-AR stimulation induces apoptosis only in a fraction of ARVMs (~15-20%) in vitro. We hypothesized that ARVMs may secrete/release a survival factor/s which protects 80-85% of cells from apoptosis. Using 2-dimensional gel electrophoresis followed by MALDI TOF and MS/MS, we identified ubiquitin in the conditioned media of ARVMs treated with ?-AR agonist, isoproterenol. Western blot analysis confirmed increased levels of ubiquitin in the conditioned media, and in the serum of mice following ?-AR stimulation. Other preliminary data suggest that extracellular ubiquitin interacts with ARVMs. Pretreatment of ARVMs with purified ubiquitin inhibited ?-AR-stimulated activation of GSK-3? and JNKs, and increases in the levels of cytosolic cytochrome C and apoptosis. Inhibition of PI3-kinase reversed the anti-apoptotic effects of extracellular ubiquitin. Anti-apoptotic effects of extracellular ubiquitin are exerted via monoubiquitination. Infusion of mice with exogenous ubiquitin inhibited ?AR-stimulated left ventricular dysfunction, cardiac myocyte apoptosis and myocardial fibrosis in mice. These observations have led to our novel hypothesis that extracellular ubiquitin plays a protective role in ?-AR-stimulated apoptosis via the activation of PI3-kinase/Akt pathway and inactivation of GSK-3?, JNKs and mitochondrial death pathway. To test this hypothesis, we will use in vitro and in vivo strategies and biochemical and proteomic approaches. Aim 1 will determine the specificity of extracellular ubiquitin in ?-AR-stimulated apoptosis using neutralizing antibodies against ubiquitin, and other apoptotic agents (angiotensin II, oxidative stress and Tumor necrosis factor-1), and test the hypothesis that secretion of ubiquitin occurs via ?2-AR-dependent signaling pathway. Aim 2 will use biochemical and proteomic approaches, and test the hypothesis that extracellular ubiquitin interacts with cellular proteins and activates PI3-kinase/Akt pathway leading to inactivation of GSK-3?, JNKs and mitochondrial death pathway, and thereby playing a protective role in ?-AR-stimulated apoptosis. Aim 3 will investigate the in vivo role of exogenous ubiquitin in ?-AR stimulated myocardial remodeling. Proposed studies investigating the role of extracellular ubiquitin in cardiac myocyte apoptosis and myocardial remodeling may uncover novel strategies for the treatment of heart failure. PUBLIC HEALTH RELEVANCE: Using proteomic and biochemical strategies, we have identified increased levels of ubiquitin in the conditioned media of adult cardiac myocytes following ?-AR stimulation, a stimulus which induces cardiac myocyte apoptosis in vitro and in vivo. Preliminary data suggest that extracellular ubiquitin interacts with cellular proteins and plays a protective role in ?AR-stimulated cardiac myocyte apoptosis. The proposed studies investigating the role of extracellular ubiquitin in cardiac myocyte apoptosis and myocardial remodeling are novel, and may uncover therapeutic potential for extracellular ubiquitin in the treatment of heart failure.

描述（由申请人提供）：交感神经活动增加是心力衰竭患者的主要特征。刺激？肾上腺素受体（β-AR），特别是β1-AR，在体外和体内增加心肌细胞凋亡，而刺激β2-AR在β-AR刺激的细胞凋亡中发挥抗细胞凋亡作用。我们已经证明，α-AR 刺激的细胞凋亡是通过糖原合成酶激酶 3 的激活介导的？ (GSK-3?) 和成年大鼠心室肌细胞 (ARVM) 中 JNK 依赖性线粒体死亡途径。在体外，β-AR 刺激仅诱导一小部分 ARVM（约 15-20%）发生细胞凋亡。我们假设 ARVM 可能会分泌/释放一种生存因子，保护 80-85% 的细胞免于凋亡。使用二维凝胶电泳，然后进行 MALDI TOF 和 MS/MS，我们鉴定了用 β-AR 激动剂异丙肾上腺素处理的 ARVM 条件培养基中的泛素。蛋白质印迹分析证实条件培养基中以及α-AR刺激后小鼠血清中的泛素水平增加。其他初步数据表明细胞外泛素与 ARVM 相互作用。用纯化的泛素预处理 ARVM 可抑制 α-AR 刺激的 GSK-3 激活？和 JNK，并增加胞质细胞色素 C 和细胞凋亡的水平。 PI3激酶的抑制逆转了细胞外泛素的抗凋亡作用。细胞外泛素的抗凋亡作用是通过单泛素化发挥的。给小鼠输注外源性泛素可抑制 βAR 刺激的小鼠左心室功能障碍、心肌细胞凋亡和心肌纤维化。这些观察结果得出了我们的新假设，即细胞外泛素通过激活 PI3 激酶/Akt 途径以及灭活 GSK-3、JNK 和线粒体死亡途径，在 β-AR 刺激的细胞凋亡中发挥保护作用。为了检验这一假设，我们将使用体外和体内策略以及生化和蛋白质组学方法。目标 1 将使用泛素中和抗体和其他凋亡剂（血管紧张素 II、氧化应激和肿瘤坏死因子-1）确定细胞外泛素在 β-AR 刺激细胞凋亡中的特异性，并检验泛素分泌通过 β2-AR 依赖性信号通路发生的假设。目标2将使用生化和蛋白质组学方法，检验细胞外泛素与细胞蛋白相互作用并激活PI3激酶/Akt通路导致GSK-3?、JNK和线粒体死亡通路失活，从而在?-AR刺激的细胞凋亡中发挥保护作用的假设。目标 3 将研究外源性泛素在 β-AR 刺激的心肌重塑中的体内作用。拟议的研究调查细胞外泛素在心肌细胞凋亡和心肌重塑中的作用，可能会揭示治疗心力衰竭的新策略。公共健康相关性：利用蛋白质组学和生化策略，我们发现在α-AR刺激后，成人心肌细胞的条件培养基中泛素水平升高，β-AR刺激是一种在体外和体内诱导心肌细胞凋亡的刺激。初步数据表明，细胞外泛素与细胞蛋白相互作用，并在βAR刺激的心肌细胞凋亡中发挥保护作用。拟议的研究细胞外泛素在心肌细胞凋亡和心肌重塑中的作用的研究是新颖的，并且可能揭示细胞外泛素在心力衰竭治疗中的治疗潜力。