Extracellular ubiquitin: role in myocyte apoptosis and myocardial remodeling

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？肾上腺素能受体？-AR)，具体来说？1-AR，体外和体内增加心肌细胞凋亡，而刺激？2-AR在？-AR-stimulated细胞凋亡。我们已经证明了？- 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jnk依赖性线粒体死亡途径在成年大鼠心室肌细胞（ARVMs）中的作用。？-AR刺激仅在一小部分arvm（~15-20%）中诱导细胞凋亡。我们假设ARVMs可能分泌/释放一种生存因子/s，保护80-85%的细胞免于凋亡。利用二维凝胶电泳、MALDI TOF和质谱联用技术，我们在经？-AR激动剂异丙肾上腺素。Western blot分析证实条件培养基中泛素水平升高，小鼠血清中泛素水平升高。基于“增大化现实”技术的刺激。其他初步数据表明细胞外泛素与arvm相互作用。纯化泛素抑制？预处理ARVMs- ar刺激GSK-3？和JNKs，胞浆细胞色素C和凋亡水平升高。抑制pi3激酶逆转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。小鼠输注外源性泛素抑制？ar刺激小鼠左心室功能障碍、心肌细胞凋亡和心肌纤维化。这些观察结果导致了我们的新假设，即细胞外泛素在？通过活化pi3 -激酶/Akt通路和灭活GSK-3？， JNKs和线粒体死亡途径。为了验证这一假设，我们将使用体外和体内策略以及生化和蛋白质组学方法。目的1将确定细胞外泛素在？使用抗泛素和其他凋亡因子（血管紧张素II、氧化应激和肿瘤坏死因子-1）的中和抗体，并测试泛素分泌通过？2- ar依赖性信号通路。Aim 2将使用生化和蛋白质组学方法，并验证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失活的假设。， JNKs和线粒体死亡途径，从而在？-AR-stimulated细胞凋亡。目的3将研究外源性泛素在？-AR刺激心肌重构。研究细胞外泛素在心肌细胞凋亡和心肌重塑中的作用可能会发现治疗心力衰竭的新策略。公共卫生相关性：使用蛋白质组学和生化策略，我们发现成人心肌细胞条件培养基中泛素水平升高。-AR刺激，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？ar刺激心肌细胞凋亡。研究细胞外泛素在心肌细胞凋亡和心肌重塑中的作用是新颖的，并可能揭示细胞外泛素在治疗心力衰竭中的治疗潜力。

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.