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

基本信息

批准号：
7835541
负责人：
KRISHNA SINGH
金额：
$ 17.88万
依托单位：
EAST TENNESSEE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-10 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7835541
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

项目摘要

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）：增加交感神经活动是心力衰竭患者的核心特征。刺激？肾上腺素能受体（？-AR），特别是？1-AR，在体外和体内增加心肌细胞凋亡，而刺激？2-AR在刺激的凋亡中扮演抗凋亡作用。我们已经表明，刺激的凋亡是通过糖原合酶激酶3的激活介导的吗？（GSK-3？）和JNK依赖性线粒体死亡途径（ARVMS）。？ - AR刺激仅在体外诱导ARVM的一部分（〜15-20％）中诱导凋亡。我们假设ARVM可以分泌/释放一个生存因子，该因子可保护80-85％的细胞免受凋亡。使用二维凝胶电泳，然后使用MALDI TOF和MS/MS，我们确定了在用？ - Ar Agonist，异丙肾上腺素的条件培养基中的泛素。 Western印迹分析证实，条件培养基中的泛素水平增加，以及随后的小鼠血清中的泛素水平。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抑制了GSK-3的激活？和JNK，并增加了胞质细胞色素C和凋亡的水平。 PI3-激酶的抑制作用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施加的。在小鼠中注入外源性泛素的小鼠抑制了AR刺激的左心室功能障碍，心肌细胞凋亡和小鼠心肌纤维化。这些观察结果导致了我们的新假设，即细胞外泛素通过激活PI3-激酶/AKT途径的激活以及GSK-3？，JNKS和线粒体死亡途径的灭活而在刺激的凋亡中起保护作用。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将使用对泛素的中和抗体和其他凋亡剂（Angiotensin II，氧化应激和肿瘤坏死因子-1）中和其他抗体的抗体来确定细胞外泛素的特异性，并测试泛素通过ubiquitin的分泌来实现？ AIM 2将使用生化和蛋白质组学方法，并测试了细胞外泛素与细胞蛋白相互作用并激活PI3-激酶/AKT途径的假设，从而导致GSK-3灭活，JNKS和MITOCHRIACHIRIAL RIALIAL DEATH途径，从而起着保护性的作用？ AIM 3将研究外源泛素在？AR刺激心肌重塑中的体内作用。提出的研究研究了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，我们已经确定了成年心脏肌细胞的条件培养基中的泛素水平提高？ - AR刺激，一种刺激，刺激会诱导心肌细胞凋亡，体外和体内。初步数据表明，细胞外泛素与细胞蛋白相互作用，并在刺激的心肌细胞凋亡中起保护作用。拟议的研究研究了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，并且可能会发现细胞外泛素在治疗心力衰竭中的治疗潜力。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Deficiency of ataxia telangiectasia mutated kinase delays inflammatory response in the heart following myocardial infarction.

DOI：
10.1161/jaha.114.001286
发表时间：
2014-12
期刊：
Journal of the American Heart Association
影响因子：
5.4
作者：
Daniel LL;Daniels CR;Harirforoosh S;Foster CR;Singh M;Singh K
通讯作者：
Singh K

Lack of ataxia telangiectasia mutated kinase induces structural and functional changes in the heart: role in β-adrenergic receptor-stimulated apoptosis.

DOI：
10.1113/expphysiol.2011.061812
发表时间：
2012-04
期刊：
Experimental physiology
影响因子：
2.7
作者：
Foster CR;Zha Q;Daniel LL;Singh M;Singh K
通讯作者：
Singh K

Β-adrenergic receptor stimulation induces endoplasmic reticulum stress in adult cardiac myocytes: role in apoptosis.

DOI：
10.1007/s11010-011-1205-7
发表时间：
2012-05
期刊：
MOLECULAR AND CELLULAR BIOCHEMISTRY
影响因子：
4.3
作者：
Dalal, Suman;Foster, Cerrone R.;Das, Bhudev C.;Singh, Mahipal;Singh, Krishna
通讯作者：
Singh, Krishna

Osteopontin: role in extracellular matrix deposition and myocardial remodeling post-MI.