Release of Tissue-specific Proteins into Coronary Perfusate as a Model for Biomarker Discovery in Myocardial Ischemia/Reperfusion Injury

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.