Wistar rats and Sprague-Dawlay (SD) male rats (SPF, 10-12 week, weight 230±10 g，n=28, respectively) were purchased from military science laboratory animal center of Chinese liberation army academy[SCXK (army), 2012-2004].The rats were divided into 8 groups according to the random number table method (7 rats per group) and named: Wistar SHAM, SD SHAM, Wistar2 hour (after myocardial infarction), SD 2h,Wistar 2 day(after myocardial infarction), SD 2d,Wistar2 week (after myocardial infarction) and SD 2w. All animals were sacrificed after experimental procedurewith potassium chloride. All experiments were conducted in accordance with the ethical requirements of Tianjin Medical University and the Third Central Hospital.
2.2 Acute Myocardial Infarction
The purchased animals were kept in the laboratory for 1 week (room temperature 25±1 ℃, each 12 hours of light and shade).Before operation, weighing and chloral hydrate (intraperitoneal, 0.3 ml / 100 g, 10%) as well as atropine (0.2 ml) were applied to anesthesia. Heart was manually exposed from the 4th intercostal space with endotracheal intubation through neck small incision aid (Fig. S1) and the left coronary artery was located, sutured and ligated at a site about 1 mm from its origin, which induced roughly 50% ischemia of the left ventricle. After ligation, observation of myocardial color changescan roughly estimate infarction area and confirm the effect of ligation at the same time. To ensure the survival rate, skilled model-making technician was needed to shorten the time of thoracotomy. Then the chest cavity of the rats was drained and when the reddish bloody fluid was extracted, stop the procedure. Intraperitoneal antibiotics were injected to prevent postoperative infection and the rats were placed in clean cages, kept separately and warm. The SHAM groups were the same as operative groups without ligation.
2.3 ECG measurement
Biopac small animal physiological instrument ECG physiological measurement module was applied to do rat real-time ECG monitoring during operation. Infarction was considered successful following a ST elevation on ECG.
2.4 Cardiac functions measurement
CO and HR were measured by PowerLab physiological measurement instrument and LabChart physiological measurement system. Two-point calibration method was adopted to measure the results of the enrolled rats. The data processing system of LabChart software was used to record the temperature of low-temperature physiological saline, injection volume, injection time, and the differences in arteriovenous blood temperature and calculate to obtain the data of the rats.
To further analyze why the tendency of 2d groups was different from other two time points, echocardiography (isoflurane, 1.5% in air) was applied to measure the diastolic and systolic volume as well as left ventricle ejection fraction (LVEF) of these two rats’ species as previously reported(9).
2.5 Animals Sample Collection
Wistar and SD rats were anesthetized at different time points, and a 10ml syringe was taken to puncture the inferior vena cava and draw blood from the inferior vena cava. The blood was drawn slowly, otherwise it was easy to cause hemolysis, which would have adverse effects on the later experiment. Then the samples were centrifuged to separate the plasma and preserved at −80°C until analysis.
2.6 Human Samples Collection
For the purposes of this prospective study, we enrolled 95 patients who were divided into four groups: the youth patient group (18–44 years old, AY group, n = 23) and elderly patient group (65–80 years old, AO group, n = 24) as well as two control groups (24 youth, SY group; 24 elderly, SO group) who were selected at the same time and within approximately the same age range after a physical examination. All patients were screened continuously from 405 patients treated from August 2013 to August 2014. The youth patients were required to provide a second blood draw during a follow‑up visit 1 year after morbidity (FY group, n = 22, one lost).Because male patients comprised a dominant proportion of acute MI (AMI) morbidity in young adults (21/23, 91.3%), we selected a matched proportion of males in the healthy youth control group, healthy elderly control group, and AMI group.
The definition of ST-elevation MI (STEMI) was established according to the third universal definition of myocardial infarction symptoms(10). Patients with the following conditions were excluded: (1) secondary to non-atherosclerotic disease,(2) history of AMI, and (3) combination with cardiac shock, infection, liver or renal insufficiency, malignant tumor, or endocrine or metabolic diseases.
Three milliliters of venous blood were drawn from the peripheral vein immediately after successful primary percutaneous coronary intervention of emergency operation, defined as the recovered blood flow of the culprit vessel to TIMI3, and preserved inan ethylene diamine tetra acetic acid anticoagulant tube. The blood was then centrifuged to separate the plasma and preserved at −80°C until analysis. All samples were collected in accordance with the ethical guidelines and written consent protocols mandated by our hospital.
2.7 Samples pretreatment
Just before analysis, these rats’ and humans’ samples were thawed at room temperature, mixed with100 µl plasma and 400 µl methanol, intensely vibrated for30 s, and incubated at 4°C for 5 min to precipitate the protein; finally, the mixture was centrifuged at 15,000 ×g for 30 min at 4°C, then the supernatant was filtrated through a 0.22 µm membrane and analyzed. Equivalent volumes of some of the fluid from each sample were mixed for quality control (QC).
2.8 Sample analysis
Chemical reagents and instruments were showed in Supplementary Table 1. Chromatography conditions were as follows. Mobile phase: Phase A: 0.1% formic acid (volume ratio); Phase B: 95%ACN and 0.1% formic acid. Chromatographic separation was performed isocratically within 15 min, and the injection volume was 10 µl. The flow rate was set at 200 µl/min. The sample manager and column oven temperature were set at4°C and 20°C, respectively. The chromatographic elutiongradient was initialized at 5% Phase B and held for 3 min. Inconsecutive 10 min periods, Phase B was gradually escalated to 50%, and then a rapid increase in Phase B to 95% was completed within 3 min. After 4 min of maintaining the high volume of organic phase gradient, Phase B was immediately reduced to 5% and this elution gradient was used to balance the analytical column in the final 4 min.
MS was operating in the positive ion mode with an ion source voltage of 4.5 kV, a capillary voltage of 30 V, cone voltage of 150 V, desolvation temperature of 275°C, sheath gas flow of 30 arb, and assistant gas flow of 5 arb (99.999% nitrogen).
Data were collected over 15 min in centroid mode over theass range 50–1000 m/z. The MS resolution was at 100,000full‑width half maximum, and the calibration standards were provided by Thermo Fisher Scientific (Caffeine,Ultramark 1621 and MRFA). MS/MS analysis was carried out with collision‑induced dissociation collision energy35 (normalization collision energy) and the collision gas was 99.999% helium.
There were 20, 23 and 22 QC samples throughout the test (equal volume mixture of each analyzed sample), respectively. Before the samples were tested, ten QC samples were analyzed continuously and there maining QC samples were inserted into the sequence after every ten samples were analyzed.
The sequence of samples was randomly generated by the Excel function before and after sample analysis (including QC), and cross‑contamination was avoided by inserting a blank between adjacent samples. The whole experiment lasted 2100 min.
Metabolic pathways and functional analysis were applied using MetaboAnalyst4.0 (The Wishart Research Group,Canada). To further analysis the feasibility of these pathways, the similarities among three groups and subgroups had been conducted.
2.9 Statistical Analysis
GraphPad Prism version 7.0 and SPSS version 23.0 were used for statistical analysis. Results are shown as mean±s.e.m. Two-tailed student's t-testing and one- or two-way ANOVA with Bonferroni post-hoc analyses were used for comparisons between different groups. A P value less than 0.05 was considered significant. Sample sizes were designed with adequate power according to the literature and our previous studies. Randomization and blinding strategy were used whenever possible.