title: Release of tissue-specific proteins into coronary perfusate as a model for biomarker discovery in myocardial ischemia/reperfusion injury.
abstract: Diagnosis of acute coronary syndromes is based on protein biomarkers, such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB) that are released into the circulation. Biomarker discovery is focused on identifying very low abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of the native protein complement hinders classical proteomic investigations. We employed an ex vivo rabbit model of myocardial ischemia/reperfusion (I/R) injury using Langendorff buffer perfusion. Nonrecirculating perfusate was collected over a temporal profile of 60 min reperfusion following brief, reversible ischemia (15 min; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis coupled to liquid chromatography and tandem mass spectrometry; geLC-MS) and gel-free (LC-MS/MS) methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples currently under investigation in large cohort studies (heart-type fatty acid binding protein; FABPH). The postischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion following acute myocardial infarction. These studies indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the presence of obfuscating plasma proteins may identify candidates for further study in humans.
authors.affiliation: School of Molecular Bioscience, The University of Sydney, and Department of Cardiology, Concord Repatriation General Hospital, New South Wales, Australia 2006. stuart.cordwell@sydney.edu.au
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Cordwell
authors.forename: Alistair V G
authors.initials: AV
authors.lastname: Edwards
authors.forename: Kiersten A
authors.initials: KA
authors.lastname: Liddy
authors.forename: Lia
authors.initials: L
authors.lastname: Moshkanbaryans
authors.forename: Nestor
authors.initials: N
authors.lastname: Solis
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Parker
authors.forename: Andy S C
authors.initials: AS
authors.lastname: Yong
authors.forename: Clement
authors.initials: C
authors.lastname: Wong
authors.forename: Leonard
authors.initials: L
authors.lastname: Kritharides
authors.forename: Brett D
authors.initials: BD
authors.lastname: Hambly
authors.forename: Melanie Y
authors.initials: MY
authors.lastname: White
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004195:Disease Models, Animal; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D060588:LIM Domain Proteins / Q000032:analysis / Q000097:blood / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis / Q000097:blood / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D009336:Necrosis / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011817:Rabbits; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D016277:Ventricular Function, Left
chemical_list: D015415:Biomarkers; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D020543:Proteome; C090114:cysteine and glycine-rich protein 3