Cardiac ischemia reperfusion (IR) injury can cause cardiac dysfunction that progresses to heart failure. One of the earliest events in this progression is thought to involve inflammatory cells and their proteases. Although beneficial at early stages after myocardial injury, excessive inflammatory reaction leads to myocyte death, scar formation, and myocardial dysfunction. We have shown that the neutrophil derived serine protease, cathepsin G (CG), induces myocyte death by anoikis that involves downregulation of proteins that maintain sarcomere integrity and contractile function including focal adhesion (FA) and cytoskeletal proteins. Little is known about specific mechanisms that maintain the integrity of FA proteins and determine their stability in cardiac myocytes. One of the primary effectors of monitoring and degrading proteins is the Ubiquitin Proteasome System (UPS). The proto-oncogene Casitas b-lineage lymphoma (Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor tyrosine kinase signaling, resulting in their ubiquitylation and proteasomal degradation. Our preliminary studies showed increased Cbl activation in response to myocardial IR injury that was associated with an increase in ubiquitylation of several proteins including FA proteins. Furthermore, we found that CG induced Cbl activation in neonatal rat cardiomyocytes in-vitro that was associated with its interaction with FAK. We also found that FAK becomes heavily serine phosphorylated prior to its inactivation and degradation in response to CG, supportive of a relationship between serine phosphorylation of proteins and their targeting to the proteasome. These data support the hypothesis that serine phosphorylation of FAK in response to CG initiates its association with Cbl, thus leading to its ubiquitylation and degradation by the UPS. These steps ultimately lead to FA signaling alteration and myocyte death by anoikis.
Aim 1 will identify the implication of serine phosphorylation in FAK turnover and myocyte anoikis.
Aim 2 will identify whether the ubiquitin ligase Cbl play a role in CG-induced FAK downregulation and myocyte anoikis.
Aim 3 will identify the signaling pathways downstream from FAK that lead to the activation of initiator caspases during CG-induced myocyte anoikis. Finally, aim 4 will determine if CG and Cbl are involved in FAK ubiquitylation/degradation and myocyte loss induced in-vivo during IR injury. This proposal seeks to link FAK serine hyper-phosphorylation in areas of inflammation to its degradation by the UPS. The fundamental new knowledge resulting from this study should identify novel targets that could diminish myocyte death and dysfunctional remodeling after myocardial IR injury.

Public Health Relevance

The research proposed in this application will delineate the role of ubiquitin ligases in focal adhesion signaling alterations caused by inflammatory proteases and will determine if serine proteases and the ubiquitin ligase Cbl could be novel targets for therapies that diminish myocyte death and dysfunctional remodeling after myocardial IR injury.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL076799-09
Application #
8277950
Study Section
Myocardial Ischemia and Metabolism Study Section (MIM)
Program Officer
Schwartz, Lisa
Project Start
2004-09-15
Project End
2013-09-17
Budget Start
2012-06-01
Budget End
2013-09-17
Support Year
9
Fiscal Year
2012
Total Cost
$371,250
Indirect Cost
$123,750
Name
Temple University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Rafiq, Khadija; Kolpakov, Mikhail A; Seqqat, Rachid et al. (2014) c-Cbl inhibition improves cardiac function and survival in response to myocardial ischemia. Circulation 129:2031-43
Libonati, Joseph R; Sabri, Abdelkarim; Xiao, Canhua et al. (2011) Exercise training improves systolic function in hypertensive myocardium. J Appl Physiol 111:1637-43
Kolwicz, Stephen C; MacDonnell, Scott M; Renna, Brian F et al. (2009) Left ventricular remodeling with exercise in hypertension. Am J Physiol Heart Circ Physiol 297:H1361-8
Kolpakov, Mikhail A; Seqqat, Rachid; Rafiq, Khadija et al. (2009) Pleiotropic effects of neutrophils on myocyte apoptosis and left ventricular remodeling during early volume overload. J Mol Cell Cardiol 47:634-45
Rafiq, Khadija; Hanscom, Marie; Valerie, Kristoffer et al. (2008) Novel mode for neutrophil protease cathepsin G-mediated signaling: membrane shedding of epidermal growth factor is required for cardiomyocyte anoikis. Circ Res 102:32-41
Sabri, Abdelkarim; Rafiq, Khadija; Seqqat, Rachid et al. (2008) Sympathetic activation causes focal adhesion signaling alteration in early compensated volume overload attributable to isolated mitral regurgitation in the dog. Circ Res 102:1127-36
Rafiq, Khadija; Kolpakov, Mikhail A; Abdelfettah, Malika et al. (2006) Role of protein-tyrosine phosphatase SHP2 in focal adhesion kinase down-regulation during neutrophil cathepsin G-induced cardiomyocytes anoikis. J Biol Chem 281:19781-92