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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HL092459-02
Application #
7835541
Study Section
Myocardial Ischemia and Metabolism Study Section (MIM)
Program Officer
Adhikari, Bishow B
Project Start
2009-05-10
Project End
2013-03-31
Budget Start
2010-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2010
Total Cost
$178,750
Indirect Cost
Name
East Tennessee State University
Department
Physiology
Type
Schools of Medicine
DUNS #
051125037
City
Johnson City
State
TN
Country
United States
Zip Code
37614
Dalal, Suman; Zha, Qinqin; Daniels, Christopher R et al. (2014) Osteopontin stimulates apoptosis in adult cardiac myocytes via the involvement of CD44 receptors, mitochondrial death pathway, and endoplasmic reticulum stress. Am J Physiol Heart Circ Physiol 306:H1182-91
Singh, Mahipal; Dalal, Suman; Singh, Krishna (2014) Osteopontin: At the cross-roads of myocyte survival and myocardial function. Life Sci 118:1-6
Daniel, Laura L; Daniels, Christopher R; Harirforoosh, Saghar et al. (2014) Deficiency of ataxia telangiectasia mutated kinase delays inflammatory response in the heart following myocardial infarction. J Am Heart Assoc 3:e001286
Steagall, Rebecca J; Daniels, Christopher R; Dalal, Suman et al. (2014) Extracellular ubiquitin increases expression of angiogenic molecules and stimulates angiogenesis in cardiac microvascular endothelial cells. Microcirculation 21:324-32
Gao, Ming; Ha, Tuanzhu; Zhang, Xia et al. (2013) The Toll-like receptor 9 ligand, CpG oligodeoxynucleotide, attenuates cardiac dysfunction in polymicrobial sepsis, involving activation of both phosphoinositide 3 kinase/Akt and extracellular-signal-related kinase signaling. J Infect Dis 207:1471-9
Foster, Cerrone R; Daniel, Laura L; Daniels, Christopher R et al. (2013) Deficiency of ataxia telangiectasia mutated kinase modulates cardiac remodeling following myocardial infarction: involvement in fibrosis and apoptosis. PLoS One 8:e83513
Steagall, R J; Sipe, A L; Williams, C A et al. (2012) Substance P release in response to cardiac ischemia from rat thoracic spinal dorsal horn is mediated by TRPV1. Neuroscience 214:106-19
Daniels, Christopher R; Foster, Cerrone R; Yakoob, Sana et al. (2012) Exogenous ubiquitin modulates chronic ýý-adrenergic receptor-stimulated myocardial remodeling: role in Akt activity and matrix metalloproteinase expression. Am J Physiol Heart Circ Physiol 303:H1459-68
Gao, Ming; Ha, Tuanzhu; Zhang, Xia et al. (2012) Toll-like receptor 3 plays a central role in cardiac dysfunction during polymicrobial sepsis. Crit Care Med 40:2390-9
Foster, Cerrone R; Zha, QinQin; Daniel, Laura L et al. (2012) Lack of ataxia telangiectasia mutated kinase induces structural and functional changes in the heart: role in ?-adrenergic receptor-stimulated apoptosis. Exp Physiol 97:506-15

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