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Time course label-free quantitative analysis of cardiac muscles of rats after myocardial infarction† Chun Li,‡a Qi Qiu,‡b Yong Wang,‡c Ping Li,‡d Cheng Xiao,e Hongxia Wang,d Yang Linb and Wei Wang*c Heart failure is a worldwide cause of mortality and morbidity and is the ultimate ending of a variety of complex diseases. This reflects our incomplete understanding of its underlying molecular mechanisms and furthermore increases the complexity of the disease. To better understand the molecular mechanisms of heart failure, we investigated dynamic proteomic differences between the heart tissue of myocardial infarction rats and the rats in the sham group at days 4, 14, 28, 45 after operation. Using a label-free quantitative proteomic approach based on nanoscale ultra-performance liquid chromatography-ESI-MSE, 133 proteins were identified at the four time points in 8 groups. 13 non-redundant proteins changed dynamically after acute myocardial infarction (AMI) in rat left ventricular (LV) tissue, including cytoskeletal proteins, metabolic enzymes, oxidative stress related proteins and ion channel proteins. The network analysis showed that the differential protein might play an important role in lipid metabolism and hypertrophic cardiomyopathy. The dynamic changes in the expression of beta-actin, alpha B-crystallin (CryAB), heat shock protein 8(HSP8), desmin and L-lactate dehydrogenase B (LDHB) were tested by the western-blot assay, and the results were consistent with the label-free quantitative proteomic results. Correlative analysis indicates that the CryAB and desmin have a better linear relation with heart function
Received 22nd September 2013, Accepted 2nd December 2013
(ejection fraction) than cardiac troponin T (cTNT). Our results provide the first experimental evidence of the
DOI: 10.1039/c3mb70422j
quantitative proteomics in vivo without ischemia-reperfusion injury or myocardial ischemia. These
proteins that are differentially expressed following myocardial infarction, using time-course label-free differential functional proteins (especially CryAB and desmin) have different patterns during the myocardial
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infarction, which may partially account for the underlying mechanisms involved in cardiac rehabilitation.
Introduction Acute myocardial infarction (AMI) resulting in heart failure is among the leading causes of morbidity and mortality in developed countries. The underlying molecular causes of cardiac dysfunction in most AMIs are still largely unknown but are expected to result from causal alterations in gene and protein expression. Proteomic technology now allows us to examine global alterations in protein expression in the diseased heart and can provide a
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China b Capital Medical University Beijing An Zhen Hospital, Beijing, China c Beijing University of Chinese Medicine, Beijing, China. E-mail: [email protected]; Tel: +86-010-64286508 d National Center of Biomedical Analysis, Beijing, China e Institute of Clinical Medicine, China-Japan Friendship Hospital, Beijing, China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3mb70422j ‡ These authors contributed equally to this work.
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new insights into the cellular mechanisms involved in cardiac dysfunction.1 Quantitative proteomics has been identified as a type of technology that will serve as a major contributor to studies aimed at uncovering disease pathways, discovering biomarkers, and providing new insights into biological processes for drug discovery.2 The methodologies for protein quantitation include 2-dimensional gel electrophoresis techniques, metabolic labeling, and stable isotope labeling methods. Recently, label-free LC-MS quantification methods have been used to determine relative abundances of proteins in cardiac samples,3–7 but these studies only included one time point at which to perform the research. In this method, peptides with a mass precision of o10 ppm and a time tolerance of o0.25 min were regarded as the same peptides, and their intensities were compared for analysis of protein amounts. The level of reproducibility in nanoUPLC allows for quantitative assessment of changes between samples with high precision without the need for stable isotope–based techniques, in particular when combined with low/high-collision energy MS analysis (MSE).8,9 MSE-based data
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acquisition permits one to collect sufficient data points in lowcollision mode to quantify peak ion intensities and, at the same time, obtain fragmentation data in high-collision mode for protein identification. The development of MSE has allowed for the collection of five to ten times more precursor ions and fragmentation data as compared to data-dependent acquisition modes.10 As such, the newly developed label-free method has sufficient sensitivity and reproducibility to meet the stability requirements of intensity, mass measurement, and retention time for label-free quantitative LC-MS measurements. In this paper, label-free LC-MS quantification methods were used, and 4 time points were chosen to monitor the dynamic changes in protein expression after AMI. A total of 133 proteins were identified and quantified in all samples, and 13 different non-redundant proteins that matched the database were analysed by network; some of them may be biomarkers of the pathological changes after acute myocardial infarction at different stages.
Materials and methods Animal care and use A total of 175 (2.5 month old) male Sprague-Dawley (SD) rats weighing 220–250 g were purchased from Vital River Laboratory Animal Center (Beijing, China). The animals were maintained under conventional conditions in our animal facility with a 12 hour light and dark cycle with access to regular food and tap water. All animal experimental protocols were approved by the Ethics Committee of Beijing University of Chinese Medicine and conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH Publication No. 85-23, revised 1996). Myocardial infarction model Rats were randomly divided into sham-operated and myocardial infarction groups (10 each). After the rats were anaesthetized with sodium pentobarbital (1%, 50 mg kg1 intraperitoneally), the trachea of each rat was intubated per-orally with a plastic tube connected to a respirator (Kent Scientific 325, China) set at a stroke volume of 3 ml kg1, a respiratory ratio of 2 : 1 and a rate of 80 strokes min1. After left thoracotomy and exposure of the heart, the left anterior descending coronary artery (LAD) was ligated with a 5–0 polypropylene suture (Yiling, Shanghai, China) directly proximal to its main branching point.11 Sham animals were treated following an identical procedure but without the tying of the polypropylene suture at the same time. The chest was then closed with 2–0 silk sutures. The intratracheal tubes were removed once the rats were breathing spontaneously, the animals were then weaned from the respirator for 3 days, and then the animals were housed 3 or 4 per cage. They were fed a standard diet and water and were maintained on a 12 hour light and dark cycle. Echocardiographic assessment of LV function Echocardiography was performed for each animal before it was euthanized using an ultrasonographic system (SIEMENS ACUSON Sequoia 512; Munich, Germany) as previously described.12
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It is used to detect the left ventricular end-systolic diameter (LVEDs), the left ventricular end-diastolic diameter (LVEDd), the ejection fraction (LVEF), fractional shortening (LVFS) and other indicators. The 15L8W-S sector scanner (14-MHz probe) was employed. The LV dimension (LVD) was measured by M-model fractional shortening (FS%) using the following equation: FS% = [(LVEDd – LVEDs)/LVEDd] 100. Heart preparation and protein extraction The heart was excised and incubated in ice-cold PBS to wash out blood, at each time point after operation (4, 14, 28, 45 days, n = 9). The myocardium in the marginal zone of the infarct region in model animals was harvested. The left ventricular myocardial below ligation bit in sham animals was also dissected. The samples were then immediately frozen in liquid nitrogen and stored at 80 1C. Frozen tissue from 9 animals of each group (from 80 1C) was ground to a powder with liquid nitrogen using a mortar and pestle. Tissue extraction medium (40 mM Tris-HCl, 7 M urea, 2 M Thiourea, 1% w/v DTT, 1 mM EDTA) (1 : 10) and protease inhibitor cocktail (1 : 50) were then added to the powder, and the mixture was ultrasonicated for 30 s. 10 ml of RNAse and 5 ml of DNAse were added, and the reaction was incubated on ice for 20 min. The samples were centrifuged at 12 000g at 4 1C for 20 min to remove the insoluble material. After centrifugation, the protein concentration of each sample was quantitated using a Bradford assay and used for following MS analysis. Protein digestion Protein digestion was performed as described.13 After adjusting the pH to 8.5 with 1 M ammonium bicarbonate, total protein extracted from each sample was chemically reduced for 45 min at 55 1C by adding DTT to a concentration of 10 mM. Next, the protein was carboxyamidomethylated in 55 mM iodoacetamide for 30 min at room temperature in the dark. Then, CaCl2 was added to 20 mM, and endoprotease Lys-C (Roche) was added to a final substrate : enzyme ratio of 100 : 1 (w/w), and the reaction was incubated at 37 1C for 12 h. The Lys-C digest was diluted to contain 1 M urea with 100 mM ammonium bicarbonate, and modified trypsin (Roche) was added to a final substrate : enzyme ratio of 50 : 1 (w/w). The trypsin digest was incubated at 37 1C for 12 h. After digestion, the peptide mixture was acidified by adding 10 ml of formic acid for further MS analysis. Samples not immediately analyzed were stored at 80 1C. Nano-UPLC-MSE analysis Nanoscale LC separation of peptides digested by Lys-C and trypsin was performed using a nanoACQUITY system (Waters, Milford, MA) equipped with a Symmetry C18 5 mm, 180 mm 20 mm pre-column and a BEH C18 1.7 mm, 75 mm 250 mm, analytical reversed-phase column (Waters), which is the same system as described by Shen et al.14 Briefly, the samples were initially transferred to the pre-column in an aqueous 0.1% formic acid solution at a flow rate of 7 ml min1 for 3 min. Mobile phase A was water with 0.1% formic acid, and mobile phase B was 0.1% formic acid in acetonitrile. The peptides were
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separated with a linear gradient of 3–40% mobile phase B over 90 min at 200 nl min1 followed by 10 min at 90% mobile phase B. The column was re-equilibrated under the initial conditions for 20 min. The column temperature was maintained at 35 1C. The lock mass was delivered from the auxiliary pump of the nanoACQUITY pump with a constant flow rate of 300 nl min1 at a concentration of 100 fmol ml1 of [Glu1] fibrinopeptide B. All samples were analyzed in triplicate. Analysis of tryptic peptides was performed using a SYNAPT HD mass spectrometer (Waters, Manchester, UK). For all measurements, the mass spectrometer was operated in v-mode with a typical resolving power of at least 10 000 full-width at halfmaximum. The TOF analyzer of the mass spectrometer was calibrated using the MS/MS fragment ions of [Glu1] fibrinopeptide B from m/z 50 to 1600. The reference sprayer was sampled with a frequency of 30 s. Accurate mass LC-MS data were collected in highdefinition MSE mode (low collision energy: 4 eV, high collision energy ramping from 15 eV to 45 eV, switching every 1.0 s, interscan time 0.02 s).10,15 The mass range was from m/z 300 to 1990. To confirm optimal column loading, all proteins present were quantified by comparison to 100 fmol of rabbit glycogen phosphorylase trypsin digest spiked into the sample.
clustered based on mass precision (typically around 5 ppm) and a retention time tolerance of o0.25 min using the clustering software included in PLGS 2.3. For protein quantification, datasets were normalized using the PLGS ‘‘auto-normalization’’ function. The basic idea of ‘‘auto-normalization’’ is that many proteins (and therefore peptide ions) won’t be changing in the experiment and so the quantitative abundance ratio should be equal to 1. Each non-changing peptide ion is regarded as a ‘‘spike’’ and this will result in potentially thousands of ‘‘spikes’’ across the dataset and these ‘‘spikes’’ are used for normalization. Included limits were all protein hits that were identified with a confidence of >95%. Only those proteins identified in at least two of three injections and with Z1.3 fold change were regarded as significantly changed. The significance of the regulation level was specified at 30%. Hence 1.3-fold (0.30 natural log scale) was used as a threshold to identify significant up- or downregulation, which is typically 2–3 times the estimated error on the intensity measurement using this system.16 All the significantly changed proteins were manually assessed by checking the matched peptide and replication level across samples.
Data processing and protein identification
Protein interactions from the Human Metabolome Database17 and protein–protein interactions in the Search Tool for the Retrieval of Interacting Genes/Proteins18 were used to construct a network containing relationships between proteins and AMIrelated genes. These proteins were connected to the 13 AMI related genes (considered at that point), allowing for 1 intermediate protein (other proteins) through STRING protein functional interaction and optimized by eliminating edges with a STRING score of 0.4 and undirected paths.19
Continuum LC-MS data were processed and searched using ProteinLynx GlobalServer version 2.3 (PLGS 2.3) (Waters Corp, Manchester, UK). Raw datasets were processed, including ion detection, deisotoping, deconvolution, and peak lists were generated based on the assignment of precursor ions and fragments based on similar retention times. The principles of the applied data clustering and normalization have been previously explained in great detail.22,24 Components are typically clustered together with a mass precision of o10 ppm and a time tolerance of o0.25 min. Alignment of elevated energy ions with low-energy precursor peptide ions was conducted with an approximate precision of 0.05 min. An NCBI Rattus database (11 653 sequences; 3 295 141 residues) downloaded from http://www.ncbi.nlm.nih.gov/sites/ was searched with data from each triplicate run with the following parameters: peptide tolerance and fragment tolerance: automatic (usually 10 ppm for peptide tolerance and 20 ppm for fragment tolerance); trypsin missed cleavages: 1; fixed modification: carbamidomethylation of cysteine; and variable modifications: N-terminal acetylation, deamidation of asparagine and glutamine, and oxidation of methionine. The rabbit glycogen phosphorylase sequence was appended to the database as an internal standard. The protein identifications were based on the detection of at least three fragment ions per peptide, with more than two peptides identified per protein. A maximum false positive rate of 4% was allowed. Quantitative analysis The analysis of quantitative changes in protein abundance, which is based on measuring peptide ion peak intensities observed in low-collision energy mode for a triplicate set, was conducted using Waters Expression, which is part of PLGS 2.3. Identical peptides from each triplicate set per sample were
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Function analysis
Western blot analysis Protein samples were prepared as described above and were subjected to Western blot analysis. The samples (50 mg) were separated by SDS-PAGE (12.5% or 15% gel) at 100 V for 2 h. Gels were then transferred to a NC membrane (the membrane was pre-soaked for 10 s in transfer buffer: 25 mM Tris, pH 7.4, 192 mM glycine, 20% methanol) at 300 mA for 1.5 h or 2 h. Each NC membrane was blocked for 2 h with 0.5% dried skim milk in TBS-T (20 mM Tris, 500 mM NaCl, 0.05% v/v Tween 20) at RT, washed three times for 15 min each with TBS-T, and then incubated with a specific primary antibody (anti-CryAB, antibeta-Actin, anti-HSP 8, anti-desmin, anti-LDHB, anti-GAPDH, abcam, USA), in TBS-T with gentle shaking overnight at 4 1C. Each membrane was washed three times for 15 min each with TBS-T and then incubated with the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG or goat anti-mouse IgG) in TBS-T with gentle shaking at 37 1C for 1 h. Each membrane was rinsed three times for 15 min each with TBS-T, developed using the Super Enhanced Chemiluminescence Detection Kit (GE healthcare, USA). All the membranes were exposed in and scanned using ChemiDox XRS + (Bio-Rad). A semiquantitative analysis based on OD was performed using Image Lab Software (Bio-Rad) and by ANOVA test analysis.
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Measurement of plasma cTNT by ELISA
Protein identification and quantitation for proteomic results
Levels of cTNT were quantified using commercial ELISA kits (Crystal Chem Inc., Downer’s Grove, USA). Each assay was performed following the related instructions. Standards at a series of concentrations were run in parallel with the samples. The concentrations in the samples were calculated in reference to the corresponding standard curves and expressed as ng ml1.
The results from all three replicates of all eight samples were combined using PLGS. After applying a replicate filter of 2 (proteins that appear in at least two out of three runs for any given sample), a P value and a minimal fold change limit, the expression results were obtained and exported to an Excel table (supplemental data, ESI†). The detailed processing is described in Methods.
Results Cardiac function related parameter After surgery, echocardiography parameters including the left ventricular end-systolic diameter (LVEDs), the left ventricular end-diastolic diameter (LVEDd), the ejection fraction (LVEF), fractional shortening (LVFS) were detected. The results show that EF and FS values in the model group are significantly different (P o 0.01). EF and FS values of ligation rats in the model group drop down by more than 50% compared with the sham-operated group, suggesting a change in weakened heart function in these models [Fig. 1 and 2]. Proteomic results of label free 133 proteins were identified and quantified in all samples [supplemental data, ESI†]. For a protein ratio of >1.3 or o0.77 as the threshold for 4 different time points protein screening, 13 non-redundant proteins were finally considered to be difference [Table 1]. Data quality assessment for the label free procedure The replication rate, intensity reproducibility, retention time (RT) reproducibility and log–log plots were used to assess the analytical reproducibility.14 Replication rate results showed that all eight samples show high rep rate 1 counts, which is normal for highly complex samples and indicates very low abundance ions detected by the algorithm that cannot be replicated. All samples show very good plots. In particular, the identified proteins in replicates 2 and 3 account for about 70%, which are very good replication rates [ESI,† Fig. S1].
Patterns of different proteins in proteome dynamics To search for patterns in the time profiles of regulated proteins, we explored clustering techniques for the dataset containing the 13 non-redundant differential proteins. The description of difference proteins in each cluster is given in Table 1, and the ratio–time Polygram according to the cluster results was drawn for a better understanding of the time-course changing expression of the difference proteins. In cluster 1, crystallin alpha B (CRYAB) and albumin were significantly up-regulated in the model group (ratio > 1.3), at day 4 after AMI, then descended and there was no significant difference between the two groups after day 14. Cluster 2 included acetyl-Coenzyme A acyltransferase 2(ACAA2), beta-actin (ACTB), desmin, heat shock protein 8(HSPA8), and the mitochondrial trifunctional protein alpha subunit (HADHA). Beta-actin, desmin, heat shock protein 8 up-regulated at day 4 after AMI, and acetyl-Coenzyme A acyltransferase 2 and the mitochondrial trifunctional protein alpha subunit were down-regulated at days 28 and/or 45 after AMI. In cluster 3, tropomyosin 1 alpha isoform b, tropomyosin 1 alpha isoform d, tropomyosin 1 alpha isoform and L-lactate dehydrogenase B (LDHB) were included, with the low expression at day 4 after AMI (ratio o 0.77), then back to the equal level with sham animals after day 14. Network analysis results for the differentially expressed proteins Results showed the network of about 13 related genes after AMI [Fig. 3], and the main functionalities are lipid metabolism and hypertrophic cardiomyopathy. In these 13 genes, ACAA2 and HADHA which are related to lipid metabolism, were reported as
Fig. 1 Cardiac function detected by echocardiography. (A) M-mode echocardiography in sham-operated at day 4 after operation. (B) M-mode echocardiography in sham-operated at 14 days after operation. (C) M-mode echocardiography in sham-operated at 28 days after operation. (D) M-mode echocardiography in sham-operated at 45 days after operation. (E) M-mode echocardiography in model group at day 4 after operation. (F) M-mode echocardiography in model group at 14 days after operation. (G) M-mode echocardiography in model group at 28 days after operation. (H) M-mode echocardiography in model group at 45 days after operation.
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Fig. 2 Echocardiography parameters include LVEF% and LVFS% in sham-operated and model groups at each time point after operation (day 4, 14, 28, 45, n = 9).
Table 1
List of differential proteins’ ratio between model and sham animals and their cluster number at each time point after operation
Description
Entry
Gene ID
Enolase 1, (alpha) isoform 1 Albumin Crystallin, alpha B, isoform CRA_a Heat shock protein 8 Beta-actin Desmin Acetyl-Coenzyme A acyltransferase 2 Mitochondrial trifunctional protein, alpha subunit Lactate dehydrogenase B Nicotinamide nucleotide transhydrogenase Tropomyosin 1, alpha isoform a Tropomyosin 1, alpha isoform b Tropomyosin 1, alpha isoform d
NP_036686.1 NP_599153.1 NP_037067.1 NP_077327.1 ABM16832.1 NP_071976.1 EDL82876.1 NP_570839.1
P04764 P02770 P23928 P63018 A1Z0K8 P48675 P13437 Q64428
mW (Da) 47098 68714 20076 70827 31726 53424 41844 82612
pI (pH)
PLGS score
Cluster 4 d (a : b)
6.1377 6.048 6.9197 5.1998 5.0808 5.0295 7.916 9.3492
346.66 1688.769 507.2087 536.4 697.22 1095.879 693.17 996.7222
1 1 1 2 2 2 2 2
0.17 0.06 0.11 0.17 0.08 0.10 0.10 0.15
1.05 1.04 0.99 1.14 1.27 1.27 0.93 0.85
0.18 0.05 0.12 0.19 0.07 0.12 0.11 0.14
1.39 1.11 1.10 1.19 0.92 1.20 0.80 0.67
0.19 0.05 0.11 0.20 0.17 0.12 0.14 0.15
45 d (g : h) 1.11 1.11 1.20 1.03 1.08 1.27 0.76 0.75
0.18 0.05 0.11 0.21 0.08 0.14 0.12 0.14
NP_036727.1 P42123 36589 5.6274 450.0005 3 NP_001013175.1 Q5BJZ3 113796 7.7932 502.1644 3
0.76 0.10 0.94 0.12 0.93 0.11 1.03 0.08 0.76 0.16 0.72 0.17 0.68 0.14 1.11 0.14
NP_001029240.1 Q63607 32835 4.5701 538.853 3 NP_001029241.1 P04692 32688 4.5141 534.9238 3 NP_001029243.1 Q6AZ25 32772 4.5042 289.72 3
0.75 0.08 1.03 0.10 1.01 0.09 1.01 0.10 0.75 0.08 1.03 0.10 1.04 0.10 1.01 0.09 0.70 0.11 0.98 0.13 1.00 0.14 0.98 0.14
novel biomarkers for the coronary heart disease.20,21 Meanwhile, LDHB, TNNT2, ACTB, CRYAB and HSPA8 were the node critical proteins which indicated the potential drug targets for AMI in our present study.
Fig. 3
1.33 1.41 1.37 1.37 2.70 1.85 0.92 1.00
14 d (c : d) 28 d (e : f)
Network analysis of 13 non-redundant proteins in heart failure rats.
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Validation of differentially expressed proteins by western blot analysis In this study, 13 proteins were identified with dynamic changes after AMI in rat LV tissue using label-free LC-MS quantification, and they can be generally divided into two pathways. In order to verify the label-free LC-MS quantification results, the node critical proteins in hypertrophic cardiomyopathy were selected to validate the results. Samples in group 2 at each time point (n = 6) were further analyzed by western blot. The specificity of the antibodies CryAB, beta-actin, heat shock protein 8, Desim and LDHB was tested by western blot using GAPDH as an internal reference [Fig. 4A]. Beta-actin is the major indicator of myocardial cytoskeletal proteins, as revealed in Fig. 4A and B, compared with sham (1.00 0.000), beta-actin (1.02 0.066) was significantly up-regulated in 4 days as well as the time prolongs in the model group (1.168 0.116, 1.223 0.128 and 1.309 0.158 in 14, 28 and 45 day respectively), with an increase of 3.02%, 44.91%, 61.13% and 74.77%. Similar to beta-actin, CryAB also has a deterioration progress as the time passes. It is up-regulated by 7.76%, 9.31%, 16.44% and 26.25% in different time course, respectively. While HSP8 is also increased in each points, but with a decreasing growth rate with 23.23%, 19.28%, 11.54% and 7.69% in 4, 14, 28 and 45 days respectively,
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Fig. 4 (A) Western blot validation of 5 proteins (n = 6 for each time point). S: sham group, M: model group. Western blot of beta-actin, crystallin alpha B, heat shock protein 8, desmin, lactate dehydrogense B and GAPDH as an internal standard. The change ratio of proteins is normalized by GAPDH based on the OD value of protein bands. (B) Graphical representations of beta-actin, crystallin alpha B and heat shock protein 8 are shown with changes in expression over time in western blot and label-free quantitive proteomic results. The result from the Western blot was consistent with the label-free quantitative proteomic result.
indicating a lasting consumption of HSP8. Interestingly, desmin has a similar pattern to HSP8, their growth rates are 25.86%, 20.22%, 15.57% and 12.68%. However, the western blot results showed that expression of LDHB protein decreased at the time of each point in the model group (decreased by 17.46%, 16.14%, 15.91% and 20.79% respectively) which is slightly different from the results of label-free; it may be due to post-translational modifications or terminal truncation. The correlative analysis showed that the growth rate of CryAB has the most close relationship with the EF value (y = 3.65x 135.97, R2 = 0.9855, P = 0.007), what’s more, desmin also showed a negative relationship with the EF value (y = 0.6645x + 51.703, R2 = 0.9525, P = 0.024); the cTNT, which was applied to diagnose the AMI in clinical practice, is also detected to compare its diagnosis sensitivity. Although it had a negative relationship with the EF value (y = 1.0035x + 1.1885, R2 = 0.9043, P = 0.048) [Table 2], but had a lower sensitivity than CryAB and desmin, indicating that CryAB and desmin may be the more sensitive biomarkers for evaluating the deterioration degree of myocardial infarction than cTNT.
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Table 2
The concentration of cTnT at different time points
4d
14 d
28 d
45 d
Sham Model Sham Model Sham Model Sham Model Mean 0.39 SD 0.165
1.24 0.201
0.47 0.189
1.17 0.277
0.45 0.079
1.11 0.238
0.47 0.273
1.08 0.262
Discussion In this study, a label-free quantitative proteomics approach was used to identify novel biomarkers of heart muscle in myocardial infarction rats and confirmed the changes in abundance levels. To study differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Proteomic tests have been developed in the rheumatology field for several years. First, 2D gel electrophoresis and more recently, SELDI–TOF-MS technology, isotope labeling and fluorescent labeling techniques have also been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results,22 as the ability to determine
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the absolute concentration of a protein (or proteins) present within a complex protein mixture is valuable for the understanding of the underlying molecular biology guiding the response to an applied perturbation.23 In previous studies, the 2-DE-MALDI-TOF and 2-DE-DIGE techniques were used to analyze the proteome of cardiac diseases, and the results showed that energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin) were altered in the hearts of ischemia- and reperfusion-injured rabbits.24 It was also noted that ramipril caused up-regulation of glutathione peroxidase, superoxide dismutase (SOD), and heart-type fatty acid binding-protein (h-FABP) and down-regulation of HSP27, while cyclophilin A was still found in the LV tissue after two months of treatment.25 Twenty-seven non-redundant identified proteins were found in LV proteins in 2-month-old CHF rats. These proteins could be categorized into 9 classes according to their functional significance: molecular chaperones (HSPs), proteins involved in endoplasmic reticulum (ER) stress and degradation pathways, oxidative stress proteins, glycolytic enzymes, fatty acid metabolism proteins, tricarboxylic acid cycle enzymes, and respiratory chain proteins.26 Twenty-three differentially expressed protein spots were separated by two-dimensional gel electrophoresis in the myocardial infarction rabbits, and 13 of them were identified by MALDI-TOF-MS at 4 weeks after myocardial ischemia by inflation or deflation, including transferrin, protein disulfide isomerase, and mitochondrial F1-ATPase.27 In this study, days 4, 14, 28, and 45 after AMI were selected in a rat model of the left ventricular infarct border zone in the model group and left ventricular cardiac in the sham group, and protein expression was compared. 3 classes including stress response-associated proteins, metabolism-associated proteins and cytoskeleton proteins were identified.
Heat shock proteins especially crystallin alpha B seem to be the better biomarkers than cTNT in CHD When cardiomyocytes are exposed to stress, production of heat shock proteins (HSPs) in the cells is enhanced. Such increases in cellular HSP production are considered to bring about tolerance against stress-induced cell damage.28 The exact role of the cellular HSPs remains unclear. In the present study, HSPs were detected in the cardiac muscle of myocardial infarction rats by label-free quantitative analysis in different levels over the time course of the experiment. HSP8 and crystallin alpha B rose at day 4 after myocardial infarction in model rats and down-regulated to the same level in sham rats at the other 3 time points. Hsp8 also plays a cardio-protective role during myocardial infarction. It was shown that NO induced HSP8 expression and delayed protection of the heart via the activities of protein kinase C by a cyclic GMP-independent pathway.29 Also, improved expression of HSP8 can partly explain the effect of ramipril in attenuating LV remodeling.30
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Crystallin alpha B, a member of the small heat shock protein family, is the most abundant low-molecular-weight heat shock protein in the heart, and recent studies have demonstrated that it plays a cardio-protective role during myocardial infarction both in vivo and in vitro.31 In addition, it was found that in addition to protecting the organization of the cytoskeletal network, the association of crystallin alpha B with actin fibers also helps to maintain the functional integrity of cells subjected to stress.32,33 It also modulates abnormal desmin aggregation and can serve a cardioprotective role.34 In this study, the up-regulation of beta-actin, desmin and crystallin alpha B at the same time point provided another evidence of the relationship among them [Fig. 4B]. Furthermore, correlation analysis indicates that crystallin alpha B has a better sensitivity for cardiac function than cTNT which is considered to be the diagnostic gold standard for CHD at present. Cytoskeleton proteins are not unchangeable and have negative correlation with the EF value After myocardial infarction, myocardial contraction associated proteins in the myocardial infarction region are broken down immediately and destroyed, resulting in reduced myocardial contractility. At this time, the infarct border zone may undergo cytoskeleton rearrangement and cardiac hypertrophy. Such pathological changes may play a compensatory role in two areas: one, in increasing myocardial contractility and in helping to maintain cardiac output, and two, in lowering chamber wall tension, decreasing myocardial oxygen consumption, and in helping to reduce the burden on the heart. However, this compensatory change shows some negative effects of cardiac hypertrophy, which can occur due to a decrease in hypoxia, energy metabolism and myocardial contractility. This will gradually weaken the compensatory cardiac function and lead to the progressive decrease of heart function. A group of abnormally expressed cytoskeletal proteins was detected in this study, which suggested that the left ventricular infarct border zone underwent changes in cardiomyocyte hypertrophy in the model group; these proteins show changes of their own in different time windows. In the early stage of myocardial infarction, the increased expression of cytoskeleton proteins, such as smooth muscle beta-actin and desmin, shows that the remodeling occurs in the normal left ventricular myocardium. The increase of actin was a kind of compensation of the decrease of myocardial contractile function, which suggested that it is inappropriate to take actin as internal reference in research studies of AMI. In addition, desmin was found in intermediate filaments, to be present in the myocardium and the smooth muscle cells. This protein is a double-stranded helical molecule with an incomplete spiral composition. In adult striated muscle, these proteins are connected to each other and form the myofibril Z line structure, even in the peripheral plasma membrane, forming a fibrous network structure. The high expression of desmin in the myocardial infarct border zone after myocardial infarction may reflect the development of left ventricular remodeling.
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Down-regulation of LDHB and ACAA2 indicates that energy metabolism disorder was compensated by fatty metabolism by beta-oxidation.
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L-Lactate
dehydrogenase B, acetyl-Coenzyme A acyltransferase 2 and different isoforms of tropomyosin 1 were down-regulated in this study. As we know, sixty to ninety percent of the energy required for cardiac events comes from fatty acid (FA) metabolism, while 10% to 40% is derived from glycolysis and lactate oxidation of pyruvate. When coronary insufficiency leads to myocardial ischemia and hypoxia, a series of metabolic changes can occur in the myocardial cells, such as blockage of fatty acid oxidation, increased anaerobic glycolysis, oxidative phosphorylation coupling, and lactic acid accumulation. L-Lactate dehydrogenase B can help pyruvate accept the hydrogen atom from NADH+H+ to reduce lactic acid concentration in the cardiac cells. In this study, down-regulation of L-lactate dehydrogenase B showed the abnormality of energy metabolism. Acetyl-Coenzyme A acyltransferase is located in the mitochondrial inner membrane and the mitochondrial matrix with fatty acid beta-oxidation activity and indirect anti-apoptosis activity. Mitochondrial trifunctional protein is one of the enzymes of the fatty acid beta-oxidation multienzyme complex, which is located in the mitochondrial inner membrane, the mitochondrial matrix and mitochondrial nucleoid, with acetylCoA C-acyltransferase activity and also respond to the insulin stimulus. The down-regulation of acetyl-Coenzyme A acyltransferase and mitochondrial trifunctional protein showed the disorder of the fatty acid metabolic process. This means that in the later stage of the AMI, fatty acid decomposed as compensatory recourse to provide the ATP for heart contraction, and fatty metabolism disorder will affect the myocardial ischemia reversely which causes the cardiac hypertrophy.
Conclusion In conclusion, the present results provide the first experimental evidence of the proteins that are differentially expressed following myocardial infarction, using time-course label-free quantitative proteomics in vivo without ischemia-reperfusion injury or myocardial ischemia. Many of these proteins deal with proliferation, growth and energy metabolism. The dynamic changes of 13 different nonredundant proteins were examined. We speculate that these functional proteins may be associated with coronary development in response to myocardial ischemia and partially account for the underlying mechanisms involved in cardiac rehabilitation. Interestingly, CryAB and desmin seemed to be the more sensitive biomarkers for evaluating the deterioration degree of myocardial infarction than cTNT. Further examination of the different expression level of one protein at different time points may provide treatment targets for myocardial infarction.
Abbreviations ACN ACAA2
Acetonitrile Acetyl-coenzyme A acyltransferase 2
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ALDH2 AMRT ANF AP APEX AST Bis BSA DTT EDTA ESI-MS FFA GAPDH Gly GSM HA HBB h-FABP LAD LC LDH MDA MI MudPIT MYH11 NADH NSAF PBS PMF SOD TEMED TF TFA Trx1
Mitochondrial aldehyde dehydrogenase 2 Accurate mass and retention time Atrial natriuretic factor Ammonium persulphate Absolute protein expression Aspartate amino transferase N,N 0 -Methylene-bis-(acrylamide) Bovine serum albumin Dithiothreitol Ethylenediamine tetraacetic acid Electrospray ionization mass spectrometry Free fatty acids Glyceraldehyde-3-phosphate dehydrogenase Glycine Gelsolin Hemagglutinin Hemoglobin B Heart-type fatty acid binding-protein Left anterior descending Liquid chromatography L-Lactate dehydrogenase Malondialdehyde Myocardial infarction Multidimensional protein identification technology Myoglobulin H11 Dehydrogenase nicotinamide adenine dinucleotidereduced dehydrogenase Normalized spectral abundance factor Phosphate-buffered saline Peptide mass fingerprint Superoxide dismutase N,N,N00 ,N00 -Tetramethyl-ethlene diamine Transferring Trifluoroacetic acid Thioredoxin 1
Sources of funding Grants from the National Department Public Benefit Research Foundation of China (no. 200807007); the Creation for Significant New Drugs Project of China (no. 2012ZX09103-201-011); The National Natural Science Foundation of China (no. 81202788) and the National Science & Technology Pillar Program (no. 2012BAI29B07).
Conflicts of interest None declared.
Acknowledgements The authors would like to thank Professor Shuoren Wang and Professor Mingjing Zhao for technical guidance on replication of the animal model. The authors were grateful to Dr Xingyun
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Chai for the suggestion of the revised paper. The authors would also like to thank the reviewers for their valuable comments. 18
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PAPER
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Time course label-free quantitative analysis of cardiac muscles of rats after myocardial infarction† Chun Li,‡a Qi Qiu,‡b Yong Wang,‡c Ping Li,‡d Cheng Xiao,e Hongxia Wang,d Yang Linb and Wei Wang*c Heart failure is a worldwide cause of mortality and morbidity and is the ultimate ending of a variety of complex diseases. This reflects our incomplete understanding of its underlying molecular mechanisms and furthermore increases the complexity of the disease. To better understand the molecular mechanisms of heart failure, we investigated dynamic proteomic differences between the heart tissue of myocardial infarction rats and the rats in the sham group at days 4, 14, 28, 45 after operation. Using a label-free quantitative proteomic approach based on nanoscale ultra-performance liquid chromatography-ESI-MSE, 133 proteins were identified at the four time points in 8 groups. 13 non-redundant proteins changed dynamically after acute myocardial infarction (AMI) in rat left ventricular (LV) tissue, including cytoskeletal proteins, metabolic enzymes, oxidative stress related proteins and ion channel proteins. The network analysis showed that the differential protein might play an important role in lipid metabolism and hypertrophic cardiomyopathy. The dynamic changes in the expression of beta-actin, alpha B-crystallin (CryAB), heat shock protein 8(HSP8), desmin and L-lactate dehydrogenase B (LDHB) were tested by the western-blot assay, and the results were consistent with the label-free quantitative proteomic results. Correlative analysis indicates that the CryAB and desmin have a better linear relation with heart function
Received 22nd September 2013, Accepted 2nd December 2013
(ejection fraction) than cardiac troponin T (cTNT). Our results provide the first experimental evidence of the
DOI: 10.1039/c3mb70422j
quantitative proteomics in vivo without ischemia-reperfusion injury or myocardial ischemia. These
proteins that are differentially expressed following myocardial infarction, using time-course label-free differential functional proteins (especially CryAB and desmin) have different patterns during the myocardial
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infarction, which may partially account for the underlying mechanisms involved in cardiac rehabilitation.
Introduction Acute myocardial infarction (AMI) resulting in heart failure is among the leading causes of morbidity and mortality in developed countries. The underlying molecular causes of cardiac dysfunction in most AMIs are still largely unknown but are expected to result from causal alterations in gene and protein expression. Proteomic technology now allows us to examine global alterations in protein expression in the diseased heart and can provide a
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China b Capital Medical University Beijing An Zhen Hospital, Beijing, China c Beijing University of Chinese Medicine, Beijing, China. E-mail: [email protected]; Tel: +86-010-64286508 d National Center of Biomedical Analysis, Beijing, China e Institute of Clinical Medicine, China-Japan Friendship Hospital, Beijing, China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3mb70422j ‡ These authors contributed equally to this work.
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new insights into the cellular mechanisms involved in cardiac dysfunction.1 Quantitative proteomics has been identified as a type of technology that will serve as a major contributor to studies aimed at uncovering disease pathways, discovering biomarkers, and providing new insights into biological processes for drug discovery.2 The methodologies for protein quantitation include 2-dimensional gel electrophoresis techniques, metabolic labeling, and stable isotope labeling methods. Recently, label-free LC-MS quantification methods have been used to determine relative abundances of proteins in cardiac samples,3–7 but these studies only included one time point at which to perform the research. In this method, peptides with a mass precision of o10 ppm and a time tolerance of o0.25 min were regarded as the same peptides, and their intensities were compared for analysis of protein amounts. The level of reproducibility in nanoUPLC allows for quantitative assessment of changes between samples with high precision without the need for stable isotope–based techniques, in particular when combined with low/high-collision energy MS analysis (MSE).8,9 MSE-based data
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acquisition permits one to collect sufficient data points in lowcollision mode to quantify peak ion intensities and, at the same time, obtain fragmentation data in high-collision mode for protein identification. The development of MSE has allowed for the collection of five to ten times more precursor ions and fragmentation data as compared to data-dependent acquisition modes.10 As such, the newly developed label-free method has sufficient sensitivity and reproducibility to meet the stability requirements of intensity, mass measurement, and retention time for label-free quantitative LC-MS measurements. In this paper, label-free LC-MS quantification methods were used, and 4 time points were chosen to monitor the dynamic changes in protein expression after AMI. A total of 133 proteins were identified and quantified in all samples, and 13 different non-redundant proteins that matched the database were analysed by network; some of them may be biomarkers of the pathological changes after acute myocardial infarction at different stages.
Materials and methods Animal care and use A total of 175 (2.5 month old) male Sprague-Dawley (SD) rats weighing 220–250 g were purchased from Vital River Laboratory Animal Center (Beijing, China). The animals were maintained under conventional conditions in our animal facility with a 12 hour light and dark cycle with access to regular food and tap water. All animal experimental protocols were approved by the Ethics Committee of Beijing University of Chinese Medicine and conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH Publication No. 85-23, revised 1996). Myocardial infarction model Rats were randomly divided into sham-operated and myocardial infarction groups (10 each). After the rats were anaesthetized with sodium pentobarbital (1%, 50 mg kg1 intraperitoneally), the trachea of each rat was intubated per-orally with a plastic tube connected to a respirator (Kent Scientific 325, China) set at a stroke volume of 3 ml kg1, a respiratory ratio of 2 : 1 and a rate of 80 strokes min1. After left thoracotomy and exposure of the heart, the left anterior descending coronary artery (LAD) was ligated with a 5–0 polypropylene suture (Yiling, Shanghai, China) directly proximal to its main branching point.11 Sham animals were treated following an identical procedure but without the tying of the polypropylene suture at the same time. The chest was then closed with 2–0 silk sutures. The intratracheal tubes were removed once the rats were breathing spontaneously, the animals were then weaned from the respirator for 3 days, and then the animals were housed 3 or 4 per cage. They were fed a standard diet and water and were maintained on a 12 hour light and dark cycle. Echocardiographic assessment of LV function Echocardiography was performed for each animal before it was euthanized using an ultrasonographic system (SIEMENS ACUSON Sequoia 512; Munich, Germany) as previously described.12
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It is used to detect the left ventricular end-systolic diameter (LVEDs), the left ventricular end-diastolic diameter (LVEDd), the ejection fraction (LVEF), fractional shortening (LVFS) and other indicators. The 15L8W-S sector scanner (14-MHz probe) was employed. The LV dimension (LVD) was measured by M-model fractional shortening (FS%) using the following equation: FS% = [(LVEDd – LVEDs)/LVEDd] 100. Heart preparation and protein extraction The heart was excised and incubated in ice-cold PBS to wash out blood, at each time point after operation (4, 14, 28, 45 days, n = 9). The myocardium in the marginal zone of the infarct region in model animals was harvested. The left ventricular myocardial below ligation bit in sham animals was also dissected. The samples were then immediately frozen in liquid nitrogen and stored at 80 1C. Frozen tissue from 9 animals of each group (from 80 1C) was ground to a powder with liquid nitrogen using a mortar and pestle. Tissue extraction medium (40 mM Tris-HCl, 7 M urea, 2 M Thiourea, 1% w/v DTT, 1 mM EDTA) (1 : 10) and protease inhibitor cocktail (1 : 50) were then added to the powder, and the mixture was ultrasonicated for 30 s. 10 ml of RNAse and 5 ml of DNAse were added, and the reaction was incubated on ice for 20 min. The samples were centrifuged at 12 000g at 4 1C for 20 min to remove the insoluble material. After centrifugation, the protein concentration of each sample was quantitated using a Bradford assay and used for following MS analysis. Protein digestion Protein digestion was performed as described.13 After adjusting the pH to 8.5 with 1 M ammonium bicarbonate, total protein extracted from each sample was chemically reduced for 45 min at 55 1C by adding DTT to a concentration of 10 mM. Next, the protein was carboxyamidomethylated in 55 mM iodoacetamide for 30 min at room temperature in the dark. Then, CaCl2 was added to 20 mM, and endoprotease Lys-C (Roche) was added to a final substrate : enzyme ratio of 100 : 1 (w/w), and the reaction was incubated at 37 1C for 12 h. The Lys-C digest was diluted to contain 1 M urea with 100 mM ammonium bicarbonate, and modified trypsin (Roche) was added to a final substrate : enzyme ratio of 50 : 1 (w/w). The trypsin digest was incubated at 37 1C for 12 h. After digestion, the peptide mixture was acidified by adding 10 ml of formic acid for further MS analysis. Samples not immediately analyzed were stored at 80 1C. Nano-UPLC-MSE analysis Nanoscale LC separation of peptides digested by Lys-C and trypsin was performed using a nanoACQUITY system (Waters, Milford, MA) equipped with a Symmetry C18 5 mm, 180 mm 20 mm pre-column and a BEH C18 1.7 mm, 75 mm 250 mm, analytical reversed-phase column (Waters), which is the same system as described by Shen et al.14 Briefly, the samples were initially transferred to the pre-column in an aqueous 0.1% formic acid solution at a flow rate of 7 ml min1 for 3 min. Mobile phase A was water with 0.1% formic acid, and mobile phase B was 0.1% formic acid in acetonitrile. The peptides were
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separated with a linear gradient of 3–40% mobile phase B over 90 min at 200 nl min1 followed by 10 min at 90% mobile phase B. The column was re-equilibrated under the initial conditions for 20 min. The column temperature was maintained at 35 1C. The lock mass was delivered from the auxiliary pump of the nanoACQUITY pump with a constant flow rate of 300 nl min1 at a concentration of 100 fmol ml1 of [Glu1] fibrinopeptide B. All samples were analyzed in triplicate. Analysis of tryptic peptides was performed using a SYNAPT HD mass spectrometer (Waters, Manchester, UK). For all measurements, the mass spectrometer was operated in v-mode with a typical resolving power of at least 10 000 full-width at halfmaximum. The TOF analyzer of the mass spectrometer was calibrated using the MS/MS fragment ions of [Glu1] fibrinopeptide B from m/z 50 to 1600. The reference sprayer was sampled with a frequency of 30 s. Accurate mass LC-MS data were collected in highdefinition MSE mode (low collision energy: 4 eV, high collision energy ramping from 15 eV to 45 eV, switching every 1.0 s, interscan time 0.02 s).10,15 The mass range was from m/z 300 to 1990. To confirm optimal column loading, all proteins present were quantified by comparison to 100 fmol of rabbit glycogen phosphorylase trypsin digest spiked into the sample.
clustered based on mass precision (typically around 5 ppm) and a retention time tolerance of o0.25 min using the clustering software included in PLGS 2.3. For protein quantification, datasets were normalized using the PLGS ‘‘auto-normalization’’ function. The basic idea of ‘‘auto-normalization’’ is that many proteins (and therefore peptide ions) won’t be changing in the experiment and so the quantitative abundance ratio should be equal to 1. Each non-changing peptide ion is regarded as a ‘‘spike’’ and this will result in potentially thousands of ‘‘spikes’’ across the dataset and these ‘‘spikes’’ are used for normalization. Included limits were all protein hits that were identified with a confidence of >95%. Only those proteins identified in at least two of three injections and with Z1.3 fold change were regarded as significantly changed. The significance of the regulation level was specified at 30%. Hence 1.3-fold (0.30 natural log scale) was used as a threshold to identify significant up- or downregulation, which is typically 2–3 times the estimated error on the intensity measurement using this system.16 All the significantly changed proteins were manually assessed by checking the matched peptide and replication level across samples.
Data processing and protein identification
Protein interactions from the Human Metabolome Database17 and protein–protein interactions in the Search Tool for the Retrieval of Interacting Genes/Proteins18 were used to construct a network containing relationships between proteins and AMIrelated genes. These proteins were connected to the 13 AMI related genes (considered at that point), allowing for 1 intermediate protein (other proteins) through STRING protein functional interaction and optimized by eliminating edges with a STRING score of 0.4 and undirected paths.19
Continuum LC-MS data were processed and searched using ProteinLynx GlobalServer version 2.3 (PLGS 2.3) (Waters Corp, Manchester, UK). Raw datasets were processed, including ion detection, deisotoping, deconvolution, and peak lists were generated based on the assignment of precursor ions and fragments based on similar retention times. The principles of the applied data clustering and normalization have been previously explained in great detail.22,24 Components are typically clustered together with a mass precision of o10 ppm and a time tolerance of o0.25 min. Alignment of elevated energy ions with low-energy precursor peptide ions was conducted with an approximate precision of 0.05 min. An NCBI Rattus database (11 653 sequences; 3 295 141 residues) downloaded from http://www.ncbi.nlm.nih.gov/sites/ was searched with data from each triplicate run with the following parameters: peptide tolerance and fragment tolerance: automatic (usually 10 ppm for peptide tolerance and 20 ppm for fragment tolerance); trypsin missed cleavages: 1; fixed modification: carbamidomethylation of cysteine; and variable modifications: N-terminal acetylation, deamidation of asparagine and glutamine, and oxidation of methionine. The rabbit glycogen phosphorylase sequence was appended to the database as an internal standard. The protein identifications were based on the detection of at least three fragment ions per peptide, with more than two peptides identified per protein. A maximum false positive rate of 4% was allowed. Quantitative analysis The analysis of quantitative changes in protein abundance, which is based on measuring peptide ion peak intensities observed in low-collision energy mode for a triplicate set, was conducted using Waters Expression, which is part of PLGS 2.3. Identical peptides from each triplicate set per sample were
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Function analysis
Western blot analysis Protein samples were prepared as described above and were subjected to Western blot analysis. The samples (50 mg) were separated by SDS-PAGE (12.5% or 15% gel) at 100 V for 2 h. Gels were then transferred to a NC membrane (the membrane was pre-soaked for 10 s in transfer buffer: 25 mM Tris, pH 7.4, 192 mM glycine, 20% methanol) at 300 mA for 1.5 h or 2 h. Each NC membrane was blocked for 2 h with 0.5% dried skim milk in TBS-T (20 mM Tris, 500 mM NaCl, 0.05% v/v Tween 20) at RT, washed three times for 15 min each with TBS-T, and then incubated with a specific primary antibody (anti-CryAB, antibeta-Actin, anti-HSP 8, anti-desmin, anti-LDHB, anti-GAPDH, abcam, USA), in TBS-T with gentle shaking overnight at 4 1C. Each membrane was washed three times for 15 min each with TBS-T and then incubated with the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG or goat anti-mouse IgG) in TBS-T with gentle shaking at 37 1C for 1 h. Each membrane was rinsed three times for 15 min each with TBS-T, developed using the Super Enhanced Chemiluminescence Detection Kit (GE healthcare, USA). All the membranes were exposed in and scanned using ChemiDox XRS + (Bio-Rad). A semiquantitative analysis based on OD was performed using Image Lab Software (Bio-Rad) and by ANOVA test analysis.
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Molecular BioSystems
Measurement of plasma cTNT by ELISA
Protein identification and quantitation for proteomic results
Levels of cTNT were quantified using commercial ELISA kits (Crystal Chem Inc., Downer’s Grove, USA). Each assay was performed following the related instructions. Standards at a series of concentrations were run in parallel with the samples. The concentrations in the samples were calculated in reference to the corresponding standard curves and expressed as ng ml1.
The results from all three replicates of all eight samples were combined using PLGS. After applying a replicate filter of 2 (proteins that appear in at least two out of three runs for any given sample), a P value and a minimal fold change limit, the expression results were obtained and exported to an Excel table (supplemental data, ESI†). The detailed processing is described in Methods.
Results Cardiac function related parameter After surgery, echocardiography parameters including the left ventricular end-systolic diameter (LVEDs), the left ventricular end-diastolic diameter (LVEDd), the ejection fraction (LVEF), fractional shortening (LVFS) were detected. The results show that EF and FS values in the model group are significantly different (P o 0.01). EF and FS values of ligation rats in the model group drop down by more than 50% compared with the sham-operated group, suggesting a change in weakened heart function in these models [Fig. 1 and 2]. Proteomic results of label free 133 proteins were identified and quantified in all samples [supplemental data, ESI†]. For a protein ratio of >1.3 or o0.77 as the threshold for 4 different time points protein screening, 13 non-redundant proteins were finally considered to be difference [Table 1]. Data quality assessment for the label free procedure The replication rate, intensity reproducibility, retention time (RT) reproducibility and log–log plots were used to assess the analytical reproducibility.14 Replication rate results showed that all eight samples show high rep rate 1 counts, which is normal for highly complex samples and indicates very low abundance ions detected by the algorithm that cannot be replicated. All samples show very good plots. In particular, the identified proteins in replicates 2 and 3 account for about 70%, which are very good replication rates [ESI,† Fig. S1].
Patterns of different proteins in proteome dynamics To search for patterns in the time profiles of regulated proteins, we explored clustering techniques for the dataset containing the 13 non-redundant differential proteins. The description of difference proteins in each cluster is given in Table 1, and the ratio–time Polygram according to the cluster results was drawn for a better understanding of the time-course changing expression of the difference proteins. In cluster 1, crystallin alpha B (CRYAB) and albumin were significantly up-regulated in the model group (ratio > 1.3), at day 4 after AMI, then descended and there was no significant difference between the two groups after day 14. Cluster 2 included acetyl-Coenzyme A acyltransferase 2(ACAA2), beta-actin (ACTB), desmin, heat shock protein 8(HSPA8), and the mitochondrial trifunctional protein alpha subunit (HADHA). Beta-actin, desmin, heat shock protein 8 up-regulated at day 4 after AMI, and acetyl-Coenzyme A acyltransferase 2 and the mitochondrial trifunctional protein alpha subunit were down-regulated at days 28 and/or 45 after AMI. In cluster 3, tropomyosin 1 alpha isoform b, tropomyosin 1 alpha isoform d, tropomyosin 1 alpha isoform and L-lactate dehydrogenase B (LDHB) were included, with the low expression at day 4 after AMI (ratio o 0.77), then back to the equal level with sham animals after day 14. Network analysis results for the differentially expressed proteins Results showed the network of about 13 related genes after AMI [Fig. 3], and the main functionalities are lipid metabolism and hypertrophic cardiomyopathy. In these 13 genes, ACAA2 and HADHA which are related to lipid metabolism, were reported as
Fig. 1 Cardiac function detected by echocardiography. (A) M-mode echocardiography in sham-operated at day 4 after operation. (B) M-mode echocardiography in sham-operated at 14 days after operation. (C) M-mode echocardiography in sham-operated at 28 days after operation. (D) M-mode echocardiography in sham-operated at 45 days after operation. (E) M-mode echocardiography in model group at day 4 after operation. (F) M-mode echocardiography in model group at 14 days after operation. (G) M-mode echocardiography in model group at 28 days after operation. (H) M-mode echocardiography in model group at 45 days after operation.
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Fig. 2 Echocardiography parameters include LVEF% and LVFS% in sham-operated and model groups at each time point after operation (day 4, 14, 28, 45, n = 9).
Table 1
List of differential proteins’ ratio between model and sham animals and their cluster number at each time point after operation
Description
Entry
Gene ID
Enolase 1, (alpha) isoform 1 Albumin Crystallin, alpha B, isoform CRA_a Heat shock protein 8 Beta-actin Desmin Acetyl-Coenzyme A acyltransferase 2 Mitochondrial trifunctional protein, alpha subunit Lactate dehydrogenase B Nicotinamide nucleotide transhydrogenase Tropomyosin 1, alpha isoform a Tropomyosin 1, alpha isoform b Tropomyosin 1, alpha isoform d
NP_036686.1 NP_599153.1 NP_037067.1 NP_077327.1 ABM16832.1 NP_071976.1 EDL82876.1 NP_570839.1
P04764 P02770 P23928 P63018 A1Z0K8 P48675 P13437 Q64428
mW (Da) 47098 68714 20076 70827 31726 53424 41844 82612
pI (pH)
PLGS score
Cluster 4 d (a : b)
6.1377 6.048 6.9197 5.1998 5.0808 5.0295 7.916 9.3492
346.66 1688.769 507.2087 536.4 697.22 1095.879 693.17 996.7222
1 1 1 2 2 2 2 2
0.17 0.06 0.11 0.17 0.08 0.10 0.10 0.15
1.05 1.04 0.99 1.14 1.27 1.27 0.93 0.85
0.18 0.05 0.12 0.19 0.07 0.12 0.11 0.14
1.39 1.11 1.10 1.19 0.92 1.20 0.80 0.67
0.19 0.05 0.11 0.20 0.17 0.12 0.14 0.15
45 d (g : h) 1.11 1.11 1.20 1.03 1.08 1.27 0.76 0.75
0.18 0.05 0.11 0.21 0.08 0.14 0.12 0.14
NP_036727.1 P42123 36589 5.6274 450.0005 3 NP_001013175.1 Q5BJZ3 113796 7.7932 502.1644 3
0.76 0.10 0.94 0.12 0.93 0.11 1.03 0.08 0.76 0.16 0.72 0.17 0.68 0.14 1.11 0.14
NP_001029240.1 Q63607 32835 4.5701 538.853 3 NP_001029241.1 P04692 32688 4.5141 534.9238 3 NP_001029243.1 Q6AZ25 32772 4.5042 289.72 3
0.75 0.08 1.03 0.10 1.01 0.09 1.01 0.10 0.75 0.08 1.03 0.10 1.04 0.10 1.01 0.09 0.70 0.11 0.98 0.13 1.00 0.14 0.98 0.14
novel biomarkers for the coronary heart disease.20,21 Meanwhile, LDHB, TNNT2, ACTB, CRYAB and HSPA8 were the node critical proteins which indicated the potential drug targets for AMI in our present study.
Fig. 3
1.33 1.41 1.37 1.37 2.70 1.85 0.92 1.00
14 d (c : d) 28 d (e : f)
Network analysis of 13 non-redundant proteins in heart failure rats.
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Validation of differentially expressed proteins by western blot analysis In this study, 13 proteins were identified with dynamic changes after AMI in rat LV tissue using label-free LC-MS quantification, and they can be generally divided into two pathways. In order to verify the label-free LC-MS quantification results, the node critical proteins in hypertrophic cardiomyopathy were selected to validate the results. Samples in group 2 at each time point (n = 6) were further analyzed by western blot. The specificity of the antibodies CryAB, beta-actin, heat shock protein 8, Desim and LDHB was tested by western blot using GAPDH as an internal reference [Fig. 4A]. Beta-actin is the major indicator of myocardial cytoskeletal proteins, as revealed in Fig. 4A and B, compared with sham (1.00 0.000), beta-actin (1.02 0.066) was significantly up-regulated in 4 days as well as the time prolongs in the model group (1.168 0.116, 1.223 0.128 and 1.309 0.158 in 14, 28 and 45 day respectively), with an increase of 3.02%, 44.91%, 61.13% and 74.77%. Similar to beta-actin, CryAB also has a deterioration progress as the time passes. It is up-regulated by 7.76%, 9.31%, 16.44% and 26.25% in different time course, respectively. While HSP8 is also increased in each points, but with a decreasing growth rate with 23.23%, 19.28%, 11.54% and 7.69% in 4, 14, 28 and 45 days respectively,
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Fig. 4 (A) Western blot validation of 5 proteins (n = 6 for each time point). S: sham group, M: model group. Western blot of beta-actin, crystallin alpha B, heat shock protein 8, desmin, lactate dehydrogense B and GAPDH as an internal standard. The change ratio of proteins is normalized by GAPDH based on the OD value of protein bands. (B) Graphical representations of beta-actin, crystallin alpha B and heat shock protein 8 are shown with changes in expression over time in western blot and label-free quantitive proteomic results. The result from the Western blot was consistent with the label-free quantitative proteomic result.
indicating a lasting consumption of HSP8. Interestingly, desmin has a similar pattern to HSP8, their growth rates are 25.86%, 20.22%, 15.57% and 12.68%. However, the western blot results showed that expression of LDHB protein decreased at the time of each point in the model group (decreased by 17.46%, 16.14%, 15.91% and 20.79% respectively) which is slightly different from the results of label-free; it may be due to post-translational modifications or terminal truncation. The correlative analysis showed that the growth rate of CryAB has the most close relationship with the EF value (y = 3.65x 135.97, R2 = 0.9855, P = 0.007), what’s more, desmin also showed a negative relationship with the EF value (y = 0.6645x + 51.703, R2 = 0.9525, P = 0.024); the cTNT, which was applied to diagnose the AMI in clinical practice, is also detected to compare its diagnosis sensitivity. Although it had a negative relationship with the EF value (y = 1.0035x + 1.1885, R2 = 0.9043, P = 0.048) [Table 2], but had a lower sensitivity than CryAB and desmin, indicating that CryAB and desmin may be the more sensitive biomarkers for evaluating the deterioration degree of myocardial infarction than cTNT.
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Table 2
The concentration of cTnT at different time points
4d
14 d
28 d
45 d
Sham Model Sham Model Sham Model Sham Model Mean 0.39 SD 0.165
1.24 0.201
0.47 0.189
1.17 0.277
0.45 0.079
1.11 0.238
0.47 0.273
1.08 0.262
Discussion In this study, a label-free quantitative proteomics approach was used to identify novel biomarkers of heart muscle in myocardial infarction rats and confirmed the changes in abundance levels. To study differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Proteomic tests have been developed in the rheumatology field for several years. First, 2D gel electrophoresis and more recently, SELDI–TOF-MS technology, isotope labeling and fluorescent labeling techniques have also been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results,22 as the ability to determine
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the absolute concentration of a protein (or proteins) present within a complex protein mixture is valuable for the understanding of the underlying molecular biology guiding the response to an applied perturbation.23 In previous studies, the 2-DE-MALDI-TOF and 2-DE-DIGE techniques were used to analyze the proteome of cardiac diseases, and the results showed that energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin) were altered in the hearts of ischemia- and reperfusion-injured rabbits.24 It was also noted that ramipril caused up-regulation of glutathione peroxidase, superoxide dismutase (SOD), and heart-type fatty acid binding-protein (h-FABP) and down-regulation of HSP27, while cyclophilin A was still found in the LV tissue after two months of treatment.25 Twenty-seven non-redundant identified proteins were found in LV proteins in 2-month-old CHF rats. These proteins could be categorized into 9 classes according to their functional significance: molecular chaperones (HSPs), proteins involved in endoplasmic reticulum (ER) stress and degradation pathways, oxidative stress proteins, glycolytic enzymes, fatty acid metabolism proteins, tricarboxylic acid cycle enzymes, and respiratory chain proteins.26 Twenty-three differentially expressed protein spots were separated by two-dimensional gel electrophoresis in the myocardial infarction rabbits, and 13 of them were identified by MALDI-TOF-MS at 4 weeks after myocardial ischemia by inflation or deflation, including transferrin, protein disulfide isomerase, and mitochondrial F1-ATPase.27 In this study, days 4, 14, 28, and 45 after AMI were selected in a rat model of the left ventricular infarct border zone in the model group and left ventricular cardiac in the sham group, and protein expression was compared. 3 classes including stress response-associated proteins, metabolism-associated proteins and cytoskeleton proteins were identified.
Heat shock proteins especially crystallin alpha B seem to be the better biomarkers than cTNT in CHD When cardiomyocytes are exposed to stress, production of heat shock proteins (HSPs) in the cells is enhanced. Such increases in cellular HSP production are considered to bring about tolerance against stress-induced cell damage.28 The exact role of the cellular HSPs remains unclear. In the present study, HSPs were detected in the cardiac muscle of myocardial infarction rats by label-free quantitative analysis in different levels over the time course of the experiment. HSP8 and crystallin alpha B rose at day 4 after myocardial infarction in model rats and down-regulated to the same level in sham rats at the other 3 time points. Hsp8 also plays a cardio-protective role during myocardial infarction. It was shown that NO induced HSP8 expression and delayed protection of the heart via the activities of protein kinase C by a cyclic GMP-independent pathway.29 Also, improved expression of HSP8 can partly explain the effect of ramipril in attenuating LV remodeling.30
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Crystallin alpha B, a member of the small heat shock protein family, is the most abundant low-molecular-weight heat shock protein in the heart, and recent studies have demonstrated that it plays a cardio-protective role during myocardial infarction both in vivo and in vitro.31 In addition, it was found that in addition to protecting the organization of the cytoskeletal network, the association of crystallin alpha B with actin fibers also helps to maintain the functional integrity of cells subjected to stress.32,33 It also modulates abnormal desmin aggregation and can serve a cardioprotective role.34 In this study, the up-regulation of beta-actin, desmin and crystallin alpha B at the same time point provided another evidence of the relationship among them [Fig. 4B]. Furthermore, correlation analysis indicates that crystallin alpha B has a better sensitivity for cardiac function than cTNT which is considered to be the diagnostic gold standard for CHD at present. Cytoskeleton proteins are not unchangeable and have negative correlation with the EF value After myocardial infarction, myocardial contraction associated proteins in the myocardial infarction region are broken down immediately and destroyed, resulting in reduced myocardial contractility. At this time, the infarct border zone may undergo cytoskeleton rearrangement and cardiac hypertrophy. Such pathological changes may play a compensatory role in two areas: one, in increasing myocardial contractility and in helping to maintain cardiac output, and two, in lowering chamber wall tension, decreasing myocardial oxygen consumption, and in helping to reduce the burden on the heart. However, this compensatory change shows some negative effects of cardiac hypertrophy, which can occur due to a decrease in hypoxia, energy metabolism and myocardial contractility. This will gradually weaken the compensatory cardiac function and lead to the progressive decrease of heart function. A group of abnormally expressed cytoskeletal proteins was detected in this study, which suggested that the left ventricular infarct border zone underwent changes in cardiomyocyte hypertrophy in the model group; these proteins show changes of their own in different time windows. In the early stage of myocardial infarction, the increased expression of cytoskeleton proteins, such as smooth muscle beta-actin and desmin, shows that the remodeling occurs in the normal left ventricular myocardium. The increase of actin was a kind of compensation of the decrease of myocardial contractile function, which suggested that it is inappropriate to take actin as internal reference in research studies of AMI. In addition, desmin was found in intermediate filaments, to be present in the myocardium and the smooth muscle cells. This protein is a double-stranded helical molecule with an incomplete spiral composition. In adult striated muscle, these proteins are connected to each other and form the myofibril Z line structure, even in the peripheral plasma membrane, forming a fibrous network structure. The high expression of desmin in the myocardial infarct border zone after myocardial infarction may reflect the development of left ventricular remodeling.
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Down-regulation of LDHB and ACAA2 indicates that energy metabolism disorder was compensated by fatty metabolism by beta-oxidation.
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L-Lactate
dehydrogenase B, acetyl-Coenzyme A acyltransferase 2 and different isoforms of tropomyosin 1 were down-regulated in this study. As we know, sixty to ninety percent of the energy required for cardiac events comes from fatty acid (FA) metabolism, while 10% to 40% is derived from glycolysis and lactate oxidation of pyruvate. When coronary insufficiency leads to myocardial ischemia and hypoxia, a series of metabolic changes can occur in the myocardial cells, such as blockage of fatty acid oxidation, increased anaerobic glycolysis, oxidative phosphorylation coupling, and lactic acid accumulation. L-Lactate dehydrogenase B can help pyruvate accept the hydrogen atom from NADH+H+ to reduce lactic acid concentration in the cardiac cells. In this study, down-regulation of L-lactate dehydrogenase B showed the abnormality of energy metabolism. Acetyl-Coenzyme A acyltransferase is located in the mitochondrial inner membrane and the mitochondrial matrix with fatty acid beta-oxidation activity and indirect anti-apoptosis activity. Mitochondrial trifunctional protein is one of the enzymes of the fatty acid beta-oxidation multienzyme complex, which is located in the mitochondrial inner membrane, the mitochondrial matrix and mitochondrial nucleoid, with acetylCoA C-acyltransferase activity and also respond to the insulin stimulus. The down-regulation of acetyl-Coenzyme A acyltransferase and mitochondrial trifunctional protein showed the disorder of the fatty acid metabolic process. This means that in the later stage of the AMI, fatty acid decomposed as compensatory recourse to provide the ATP for heart contraction, and fatty metabolism disorder will affect the myocardial ischemia reversely which causes the cardiac hypertrophy.
Conclusion In conclusion, the present results provide the first experimental evidence of the proteins that are differentially expressed following myocardial infarction, using time-course label-free quantitative proteomics in vivo without ischemia-reperfusion injury or myocardial ischemia. Many of these proteins deal with proliferation, growth and energy metabolism. The dynamic changes of 13 different nonredundant proteins were examined. We speculate that these functional proteins may be associated with coronary development in response to myocardial ischemia and partially account for the underlying mechanisms involved in cardiac rehabilitation. Interestingly, CryAB and desmin seemed to be the more sensitive biomarkers for evaluating the deterioration degree of myocardial infarction than cTNT. Further examination of the different expression level of one protein at different time points may provide treatment targets for myocardial infarction.
Abbreviations ACN ACAA2
Acetonitrile Acetyl-coenzyme A acyltransferase 2
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ALDH2 AMRT ANF AP APEX AST Bis BSA DTT EDTA ESI-MS FFA GAPDH Gly GSM HA HBB h-FABP LAD LC LDH MDA MI MudPIT MYH11 NADH NSAF PBS PMF SOD TEMED TF TFA Trx1
Mitochondrial aldehyde dehydrogenase 2 Accurate mass and retention time Atrial natriuretic factor Ammonium persulphate Absolute protein expression Aspartate amino transferase N,N 0 -Methylene-bis-(acrylamide) Bovine serum albumin Dithiothreitol Ethylenediamine tetraacetic acid Electrospray ionization mass spectrometry Free fatty acids Glyceraldehyde-3-phosphate dehydrogenase Glycine Gelsolin Hemagglutinin Hemoglobin B Heart-type fatty acid binding-protein Left anterior descending Liquid chromatography L-Lactate dehydrogenase Malondialdehyde Myocardial infarction Multidimensional protein identification technology Myoglobulin H11 Dehydrogenase nicotinamide adenine dinucleotidereduced dehydrogenase Normalized spectral abundance factor Phosphate-buffered saline Peptide mass fingerprint Superoxide dismutase N,N,N00 ,N00 -Tetramethyl-ethlene diamine Transferring Trifluoroacetic acid Thioredoxin 1
Sources of funding Grants from the National Department Public Benefit Research Foundation of China (no. 200807007); the Creation for Significant New Drugs Project of China (no. 2012ZX09103-201-011); The National Natural Science Foundation of China (no. 81202788) and the National Science & Technology Pillar Program (no. 2012BAI29B07).
Conflicts of interest None declared.
Acknowledgements The authors would like to thank Professor Shuoren Wang and Professor Mingjing Zhao for technical guidance on replication of the animal model. The authors were grateful to Dr Xingyun
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Chai for the suggestion of the revised paper. The authors would also like to thank the reviewers for their valuable comments. 18
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