Proteomic analysis of effluents from perfused human heart for transplantation: identification of potential biomarkers for ischemic heart damage
© Li et al; licensee BioMed Central Ltd. 2012
Received: 31 October 2011
Accepted: 23 March 2012
Published: 23 March 2012
Biomarkers released from the heart at early stage of ischemia are very important to diagnosis of ischemic heart disease and salvage myocytes from death. Known specific markers for blood tests including CK-MB, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are released after the onset of significant necrosis instead of early ischemia. Thus, they are not good biomarkers to diagnose myocardial injury before necrosis happens. Therefore, in this study, we performed proteomic analysis on effluents from perfused human hearts of donors at different ischemic time.
After global ischemia for 0 min, 30 min and 60 min at 4°C, effluents from five perfused hearts were analyzed respectively, by High performance liquid chromatography-Chip-Mass spectrometry (HPLC-Chip-MS) system. Total 196 highly reliable proteins were identified. 107 proteins were identified at the beginning of ischemia, 174 and 175 proteins at ischemic 30 min and ischemic 60 min, respectively. With the exception of cardiac troponin I and T, all known biomarkers for myocardial ischemia were detected in our study. However, there were four glycolytic enzymes and two targets of matrix metalloproteinase released significantly from the heart when ischemic time was increasing. These proteins were L-lactate dehydrogenase B(LDHB), glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate isomerase (GPI), phosphoglycerate mutase 2 (PGAM2), gelsolin and isoform 8 of titin. PGAM2, LDHB and titin were measured with enzyme-linked immunosorbent assays kits. The mean concentrations of LDHB and PGAM2 in samples showed an increasing trend when ischemic time was extending. In addition, 33% identified proteins are involved in metabolism. Protein to protein interaction network analysis showed glycolytic enzymes, such as isoform alpha-enolase of alpha-enolase, isoform 1 of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, had more connections than other proteins in myocardial metabolism during ischemia.
It is the first time to use effluents of human perfused heart to study the proteins released during myocardial ischemia by HPLC-Chip-MS system. There might be many potential biomarkers for mild ischemic injury in myocardium, especially isoform 8 of titin and M-type of PGAM2 that are more specific in the cardiac tissue than in the others. Furthermore, glycolysis is one of the important conversions during early ischemia in myocardium. This finding may provide new insight into pathology and biology of myocardial ischemia, and potential diagnostic and therapeutic biomarkers.
KeywordsProteome analysis Human perfused heart Ischemia
Ischemic Heart Disease (IHD) is the most common cause of death and a major cause of hospital admissions in most Western countries . The diagnosis of IHD is based on particular symptoms, an electrocardiogram, an X-ray of the chest and blood tests. Reasonably specific markers for blood tests including creatine kinase muscle/brain isoform (CK-MB), cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are released after the onset of significant necrosis instead of early ischemia, and they all require a level of myocardial necrosis to prompt their release from myocytes before they can be detected. Those biomarkers are impossible to be detected at early stage of ischemia and the diagnosis of IHD is often ambiguous. On the other hand, it is impossible to salvage dead myocytes at the stage of necrosis when specific markers such as CK-MB, cTnT and cTnI are detected. Thus, biomarkers detectable before the onset of significant necrosis would be more important than those in current use. Evidence has shown that markers released upon initiation of ischemia alone may exist [2–4]. If it is true, such markers would offer the opportunity for early diagnosis of IHD before permanent myocyte damage occurs, which will allow possible salvage of the myocardium by timely reperfusion. However, it is very difficult to discover a novel biomarker by screening the entire proteome of plasma from the IHD patients [4, 5]. This is because there are many highly abundant proteins present in the serum or plasma, which will mask biomarkers, presumably the less abundant proteins. Proteomic analysis of the plasma from patients with acute coronary syndromes (ACS) revealed only five differentially expressed proteins, all of which were highly abundant plasma proteins . Thus, it is very important to eliminate the interference by highly abundant plasma proteins. Isolated perfused heart effluent is a novel model for protein biomarker discovery. This model dispenses with most highly abundant proteins in blood . In our study, effluents from human perfused heart for transplantation at different ischemic time points were collected for proteomic analysis to identify potential ischemic biomarkers.
Materials and methods
Effluent collection and concentration
Clinical information of the five heart donors
The operation of the donor heart dissection was performed with a standard procedure. Briefly, the patients were anesthetized with isoflurane, intubated and ventilated with 100% oxygen. The heart and great vessels were exposed after sternotomy. Heparin at 3 mg/kg was injected intravenously for systemic anticoagulation. After a cardioplegic cannula was placed into the ascending aorta, one liter of a hypothermic (4°C) hyperkalemic crystalloid solution (composition in mmol/L: Na + 127, K + 20, Mg2+ 8, Cl- 20, SO4 - 8, HCO3 - 20, pH 7.9) was infused into the aortic root to achieve cardioplegic arrest. After cardioplegic infusion, the donor heart was extracted and placed in a basin containing 2000 ml cardioplegia solution at 4°C, The cardioplegic cannula and aortic cross-clamp were left in place to permit perfusion. Then, hypothermic cardioplegia was infused continually through the cardioplegic cannula until there was no blood in the effluent judged by eyes. A catheter was placed from inferior vena entrance into the exit of coronary sinus. The catheter was fixed and these open ends of superior and inferior vena, pulmonary arteries and veins were clamped. 200 ml hypothermic cardioplegia was infused into aortic root within 2 min and the effluent from the coronary sinus was collected into the 50 ml polypropylene tubes as the sample for the first time point. Finally, the heart was placed in a bag, and stored on ice. After 30-min ischemia, 200 ml cold cardioplegia was infused through the cardioplegic cannula, effluent was collected for the second time point. After ischemia for 60 min, effluent was collected again as the sample for the third time point. The effluent samples from each of the 5 hearts were mixed with protease inhibitors (1.67 mL of 100 mM NaN3, 2.5 mL of 11.5 mM PMSF) to avoid proteolysis and then cell debris and insoluble solids were removed by centrifugation at 10 000 × g for 15 min at 4°C. The precipitates were removed and then the supernatants were concentrated by using the Amicon Ultra-15 3 kDa MWCO centrifugal filters (Millipore, USA). Finally, the protein was stored at -80°C for further analysis.
Removal of High-Abundance Proteins
All procedures of removing high-abundance proteins were performed according to manufacturer's instructions. Briefly, each effluent sample was thawed and centrifuged at 10 000 × g for 30 min at 4°C. Multiple affinity removal system 4.6 × 50 Hu-7 (5188-6409, Agilent Technologies) was used to remove high-abundance proteins from the effluent samples according to a standard liquid chromatography protocol. Briefly, the sample was diluted three times with Buffer A and centrifuged at 16 000 g for 10 min. The diluted sample was used for injection at a flow rate of 0.25 ml/min. The low-abundance protein fraction from 1.5 min to 4.5 min was collected into a 1.5 ml Eppendorf tube and store at 4°C for further analysis, and the high-abundance protein fraction was eluted with Buffer B at a flow rate of 1.0 ml/min, then the column was regenerated with buffer A.
Protein concentrating and desalting
The low-abundance proteins were then concentrated and desalted by using the Amicon Ultra-15 10 kDa MWCO centrifugal filters (Millipore) according to manufacturer's instructions. Briefly, 10 ml of low-abundance fraction was filled into five spin concentrators with 10 kDa molecular weight cutoff. The samples were spun at 14 000 g for 30 min at 4°C, the filtrate was discarded, the concentrated protein was kept, and then the concentrator was refilled with an appropriate buffer (8 M urea) and spun again. After repeating this procedure three times, the sample was collected. Protein content was determined by using Bradford assay kit (Thermo, USA) and the sample was aliquoted into 40 ug fractions.
In-solution tryptic digestion
In-solution tryptic digestion was performed according to the standard protocol provided by Agilent Technologies. Briefly, the Eppendorf tube contained 40 ug protein was added to 10 μl of 10 mM dithiothreitol (DTT) and incubated for 1 h at 56°C. After 20 μl of 20 mM iodoacetamide (IAM) was added, the Eppendorf tube was placed in dark for 1 h at room temperature. Then, 10 μl of 10 mM DTT was added again to quench the excess IAM for 1 h at 37°C. Protein was digested by 40 μl trypsin (12.5 ng/μL) (Sequencing Grade, Promega, USA) for 12 h at 37°C. Finally, the reaction was terminated by adding 2.5 μl formic acid.
High performance liquid chromatography-Chip-Mass spectrometry (HPLC-Chip-MS) system analysis
The sample was analyzed on an Agilent 1200 HPLC and 6330 Ion Trap system (Agilent technologies, USA) as described previously [8–10]. 1 μl of digests (400 ng) were injected on a Zorbax chip composed of an enrichment column (560.3 mm, 5 mm particles) and a Zorbax 300SB C18 (75 μm × 150 mm, 3.5 mm particles) analytical column. The mobile phase for both capillary pump and nanopump consisted of 0.1% formic acid in distilled water (A) and 0.1% formic acid in 90/10 acetonitrile/distilled water (B). The flow rate for the capillary pump was held constant at 4 μl/min in 3% B (isocratic) while the flow rate for the nanopump was 0.3 μl/min, following a gradient of 3 - 75% B in 70 min. The mass spectrum was operated in chip positive ionization mode, with voltage at 4 KV, drying gas temperature at 325°C and drying gas flow at 6 L/min. In-source voltage was set at 1850 V, capillary exit at 96.4 V, skimmer at 40 V, end plate offset at -500 V. Automatic MS/MS in a data-dependent manner was acquired in enhanced mode at m/z 200 - 1600. Due to statistical fluctuations of peptide precursor selection during MS/MS acquisition, three LC-MS/MS assays were run with each sample in order to be able to do a proper proteomic comparison.
Enzyme-linked immunosorbent assays (ELISA)
The concentration of three interested proteins identified by MS was quantitatively determinated with phosphoglycerate mutase 2(PGAM2) human ELISA kit (Cusabio biotech CO., LTD, China), L-lactate dehydrogenase B(LDHB) human ELISA kit and titin human ELISA kit (Uscn Life Science Inc, China) according to the manufacturer's instructions in a blinded manner. Optical densities were measured at 450 nm by an eight-channel spectrophotometer. The concentration of LDHB, PGAM2 and titin was calculated using respective assay standard curves. The results were represented as mean ± SD.
Data analysis and statistics
The MS/MS data were searched automatically against the international protein index (IPI)human database  using the Spectrum Mill Proteomics Workbench software (RevA.03.03, Agilent, USA). Only peptides with Spectrum Mill score more than 8 and Spectrum Mill Scored Peak Intensity(SPI) > 70% were considered positives. The confidence of all identified proteins must have more than 95%. In addition, protein with p-value < 0.05 was considered as significant. After each protein was identified, the relative abundance of protein in the sample was quantified by recording the mean of the peak intensities(MPI) of the component peptides . The quantitative results were analyzed by one way analysis of variance followed by the Tukey test after confirmation of normal distribution of the data (data are presented as means ± SD) or by Kruskal-Wallis analysis of variance on ranks followed by the Dunn's test when the data are not normally distributed. A P ≤ 0.05 was accepted as significant. All statistical analyses were performed via the SigmaStat software (Systat Software, Inc., Point Richmond, CA) . Gene Ontology (GO) analysis was done by using Expression Analysis Systematic Explorer (EASE) software . A protein-protein interaction network on metabolism was done by downloading pathway data from Kyoto Encyclopedia of Genes and Genomes(KEGG) database, and then enzyme-enzyme interaction (ECrel) and protein-protein interaction (PPrel) was analyzed by KEGGSOAP software . Finally, the network of relationship between protein to protein was built and brought forth by Medusa software . The network was graphically visualized as nodes (proteins) and edges (the relationships between proteins).
Results and discussion
Clinical data and high-abundant protein removal
Protein identification by HPLC-Chip-MS system
Part of the identified proteins that may be interesting in potential biomarker discovery of early ischemia in myocardium
Isoform M1 of Pyruvate kinase isozymes M1/M2
Actin, alpha cardiac muscle 1
Aspartate aminotransferase 1
metabolism (amino acid)
Creatine kinase M-type
Heart -fatty acid-binding protein,
L-lactate dehydrogenase B chain
Isoform 1 of Triosephosphate isomerase
Isoform 8 of Titin
Isoform 1 of Gelsolin
Phosphoglycerate mutase 2
Adipocyte -fatty acid-binding protein
Adenylate kinase 1
Creatine kinase B-type
Four and a half LIM domains 1 variant
Malate dehydrogenase, cytoplasmic
Phosphatidylethanolamine-binding protein 1
PGM1 65 kDa protein
magnesium ion binding
Comfirmation of protein expression by ELISA
Gene network analysis for metabolism
In this study, we analyzed low-abundance proteins from effluents of perfused human hearts after mild ischemic injury by HPLC-Chip-MS system. Many known clinic markers for myocardial ischemia, proteins specific to heart and glycolytic enzymes were among the proteins identified. Among them, four glycolytic enzymes, L-LDH, GAPDH, GPI, PGAM2, and two targets of MMP, gelsolin and isoform 8 of titin, are significantly released into the effluent during myocardial ischemia. They are potential biomarkers for mild ischemic injury in myocardium, especially isoform 8 of titin and M-type of PGAM2, which are more specific for cardiac tissue. Furthermore, the results of gene network analysis for metabolism show glycolysis is one of the important conversions during early ischemia in myocardium. This finding may provide new insight into pathology and biology of myocardial ischemia, and potential diagnostic and therapeutic biomarkers.
Ischemic heart disease
Creatine kinase muscle/brain isoform
Creatine kinase M-type
Creatine kinase B-type
Cardiac troponin T
Cardiac troponin I
Acute coronary syndromes
- pI :
Molecular weight cutoff
High performance liquid chromatography
International protein index
Scored peak intensity
Expression analysis systematic explorer
Lactate dehydrogenase B
Isoform M1 of pyruvate kinase isozymes M1/M2
Heart-type fatty acid-binding protein
Isoform 1 of triosephosphate isomerase
Phosphoglycerate mutase 2
Isoform alpha-enolase of alpha-enolase
False discovery rate
Enzyme-linked immunosorbent assays
Kyoto encyclopedia of genes and genomes
Mean of the peak intensities.
This work was supported by the National Natural Science Foundation of China (Grant No. 30871061 and No. 81070094) and the Natural Science Foundation Project of Chongqing(CSTC,2009BA5016). We thank Professor Zhiyi Zuo (the university of Virginia, USA) for his careful revision of the manuscript and Shanghai SensiChip Tech&infor Company for the assistance with bioinformatics.
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