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（？具体来说，？1-AR，增加心肌细胞凋亡在体外和体内，而刺激？2-AR在？AR刺激的细胞凋亡。我们已经证明了吗？- AR刺激的细胞凋亡是通过激活糖原合成酶激酶-3介导的？（GSK-3？）和JNK依赖的线粒体死亡途径在成年大鼠心室肌细胞（ARVMs）。?- AR刺激在体外仅在一部分ARVM（~15-20%）中诱导凋亡。我们假设ARVM可能分泌/释放一种保护80-85%细胞免于凋亡的存活因子。利用二维凝胶电泳，然后进行MALDI TOF和MS/MS，我们鉴定了用？- AR激动剂，异丙肾上腺素。蛋白质印迹分析证实，在条件培养基中，以及在小鼠血清中，AR刺激。其他初步数据表明，细胞外泛素与ARVM相互作用。用纯化的泛素抑制剂预处理ARVM？- AR刺激GSK-3的激活？和JNK，以及细胞溶质细胞色素C和细胞凋亡水平的增加。抑制PI 3-激酶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通过单泛素化发挥。输注外源性泛素的小鼠是否受到抑制？AR刺激小鼠左心室功能不全、心肌细胞凋亡和心肌纤维化。这些观察结果使我们提出了一个新的假设，即细胞外泛素在？AR通过激活PI 3-激酶/Akt通路和失活GSK-3？刺激凋亡，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物化学和蛋白质组学方法。目的1将确定细胞外泛素的特异性？AR刺激的细胞凋亡，使用中和抗体对泛素，和其他凋亡剂（血管紧张素II，氧化应激和肿瘤坏死因子-1），并测试假设，分泌泛素发生通过？2-AR依赖性信号通路。目的2将利用生物化学和蛋白质组学方法，验证细胞外泛素与细胞内蛋白质相互作用，激活PI 3-kinase/Akt通路，导致GSK-3？失活的假说，JNKs和线粒体死亡途径，从而发挥保护作用？AR刺激的细胞凋亡。目的3：研究外源性泛素在？AR刺激心肌重塑。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为心力衰竭的治疗提供新的策略。公共卫生相关性：利用蛋白质组学和生物化学的策略，我们已经确定了增加水平的泛素在成人心肌细胞的条件培养基以下？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，并发挥保护作用？AR刺激的心肌细胞凋亡。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的，并可能揭示细胞外泛素在心力衰竭治疗中的治疗潜力。

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.