title: Quantitative profiling of the rat heart myoblast secretome reveals differential responses to hypoxia and re-oxygenation stress.
abstract: Secretion of bioactive mediators regulates cell interactions with the microenvironment in tissue homeostasis and wound healing processes. We assessed the cardiomyocyte secretory response to hypoxia with the aim of identifying key mediators of tissue pathology and repair after ischemic heart attack. We profiled the secretome of rat H9C2 cardiomyoblast cells subjected to 16h hypoxia followed by 24h re-oxygenation using iTRAQ and label-free quantitative proteomics. A total of 860 and 2007 proteins were identified in the iTRAQ and label-free experiments respectively. Among these proteins, 1363 were identified as being secreted proteins, including mediators of critical cellular functions that were modulated by hypoxia/re-oxygenation stress (SerpinH1, Ppia, Attractin, EMC1, Postn, Thbs1, Timp1, Stip1, Robo2, Fat1). Further analysis indicated that hypoxia is associated with angiogenesis, inflammation, and remodeling of the extracellular matrix (ECM), whereas subsequent re-oxygenation was instead associated with modified secretion of proteins involved in suppression of inflammation, ECM modification, and decreased output of anti-apoptosis proteins. These data indicate that hypoxia and subsequent re-oxygenation modify the cardiomyocyte secretome in order to mitigate cellular injury and promote healing. The identified changes in cardiomyocyte secretome advance our current understanding of cardiac biology in ischemia/reperfusion injury and may lead to the identification of novel prognostic biomarker.
  
  
  CONCLUSIONS
  Cardiovascular diseases (CVDs) are the leading cause of death globally. Myocardial infarction (MI) resulting from ischemic heart disease represents a substantial component of CVD-associated mortality, and is associated with obstruction of blood flow to the myocardium. Restoration of blood flow through the occluded coronary artery is the current most effective therapy to limit infarct size and preserve cardiac function after acute myocardial infarction. However, this treatment does not prevent subsequent development of heart failure in some patients. Reperfusion following ischemia causes additional cell death and increase in infarct size, a phenomenon called myocardial ischemia/reperfusion (I/R) injury. In order to advance our current understanding of cardiac biology in ischemia/reperfusion injury, we assessed the cardiomyocyte secretory response to hypoxia with the aim of identifying key mediators of tissue pathology and repair after ischemic heart attack. We profiled the secretome of rat H9C2 cardiomyoblast cells subjected to 16h hypoxia followed by 24h re-oxygenation using LC-MS/MS-based iTRAQ and label-free quantitative proteomics approaches. We identified many secreted proteins as mediators of critical cellular functions that were modulated by hypoxia and re-oxygenation stress. Further analysis of these modulated secretory proteins indicated that hypoxia is associated with angiogenesis, inflammation, and remodeling of the extracellular matrix (ECM), whereas subsequent re-oxygenation/reperfusion was instead associated with modified secretion of proteins involved in suppression of inflammation, ECM modification, and decreased output of anti-apoptosis proteins. These data indicate that hypoxia and subsequent re-oxygenation modify the cardiomyocyte secretome in order to mitigate cellular injury and promote healing. The identified changes in cardiomyocyte secretome may lead to the identification of novel prognostic biomarkers secreted from injured heart tissues into the circulation of patients with cardiovascular disease.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ren
authors.affiliation: National University Heart Centre, Department of Cardiac, Thoracic & Vascular Surgery, Singapore 119228, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Vitaly
authors.initials: V
authors.lastname: Sorokin
authors.affiliation: National University Heart Centre, Department of Cardiac, Thoracic & Vascular Surgery, Singapore 119228, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Kian Keong
authors.initials: KK
authors.lastname: Poh
authors.affiliation: Department of Cardiology, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433, Singapore.
authors.forename: Hee Hwa
authors.initials: HH
authors.lastname: Ho
authors.affiliation: National University Heart Centre, Department of Cardiac, Thoracic & Vascular Surgery, Singapore 119228, Singapore; Department of Surgery, Yong Loo Lin School of Medicine, Singapore 119228, Singapore.
authors.forename: Chuen Neng
authors.initials: CN
authors.lastname: Lee
authors.affiliation: Department of Surgery, Yong Loo Lin School of Medicine, Singapore 119228, Singapore; Experimental Cardiology Laboratory, Cardiology, University Medical Center Utrecht, the Netherlands; Interuniversity Cardiovascular Institute of the Netherlands, Utrecht, the Netherlands.
authors.forename: Dominique
authors.initials: D
authors.lastname: de Kleijn
authors.affiliation: Institute of Medical Biology, 8A Biomedical Grove, #05-505 Immunos, Singapore 138648, Singapore.
authors.forename: Sai Kiang
authors.initials: SK
authors.lastname: Lim
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors.forename: James P
authors.initials: JP
authors.lastname: Tam
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore. Electronic address: sksze@ntu.edu.sg.
authors.forename: Siu Kwan
authors.initials: SK
authors.lastname: Sze
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D009124:Muscle Proteins / Q000378:metabolism*; D032386:Myoblasts, Cardiac / Q000378:metabolism* / Q000473:pathology; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D020543:Proteome / Q000378:metabolism*; D051381:Rats
chemical_list: D015415:Biomarkers; D009124:Muscle Proteins; D020543:Proteome
keywords: Cardiac; H9C2 cell; Hypoxia; I/R injury; Quantitative proteomics; Secretome