TAC model
All animal experiments comply with the ARRIVE guidelines and were approved by the Ethics Committee of Animal Welfare at the Medical Centers of Chongqing Medical University (Chongqing, China) and Shenzhen University General Hospital (Guangdong, China).
Male Sprague-Dawley (SD) rats (6 weeks old, body weight 200g±20 g) were provided by the Experimental Animal Center of Chongqing Medical University and housed in the individually ventilated cage (IVC) facility at the Experimental Animal Center of Chongqing Medical University. Left ventricular remodeling and heart failure were induced by TAC as previously described [10]. Briefly, the animals were anesthetized by a single intraperitoneal injection of pentobarbital (60 mg/kg) and then placed on a heating pad of 37 ℃ to maintain body temperature. A small incision was made in the left third intercostal space, and artificial ventilation (Harvard Apparatus, USA) was set to allow direct access to the thorax. After identification of the transverse aorta, a 16-gauge needle (O.D. 1.6 mm) was placed between the right innominate artery and left common carotid artery. Aortic constriction was then performed by ligating the aorta with a 2-0 silk. The needle was removed rapidly after the aorta was tied tightly. The chest cavity and skin were stitched by a 6-0 polypropylene suture (Prolene, Ethicon). After approximately 10 more min of ventilation, when spontaneous breathing was restored, the rat was extubated and returned to the housing facility for maintenance.
Forty SD rats were randomly conducted the TAC or sham surgery. The sham group underwent the same surgical procedures as the TAC group but without ligation of the aortic arch. One animal in the sham group and one animal in the TAC group died of bleeding during the surgery. One animal in the sham group died of pneumothorax during the surgery. Finally, 18 animals in the sham group and 19 animals in the TAC group survived and were used for the experiment. The TAC rats were subjected to further examinations at 0 week (0W, n=6), 3 weeks (3 W, n=6) and 9 weeks (9 W, n=7) respectively after surgery. Heart rate and blood pressure were assessed by measuring parameters such as blood flow, blood pressure and pulse at the base of the tail using a rat tail-cuff blood pressure system.
Echocardiography
Transthoracic echocardiography was performed after TAC surgery to examine the changes in cardiac function and remodeling. The rats were anesthetized by a single intraperitoneal injection of pentobarbital (60 mg/kg), and echocardiography was then performed with VeVo2100 (VisualSonics, Canada). The diastolic intraventricular septum, LV end diastolic diameter, diastolic posterior wall thickness, LV internal dimension in systole, and percent LV fractional shortening were assessed from M-mode images, as previously described [11].
Cardiac catheter
Hemodynamic measurements were performed at 0 W, 3 W and 9 W after the surgery, following a reported method [12]. Briefly, the rats were anesthetized by a single intraperitoneal injection of pentobarbital (60 mg/kg) and then placed on a controlled heating table. The left carotid artery and right jugular vein were both cannulated with two fluid-filled polyethylene catheters that were connected to pressure transducers. Along with the catheters inserted into the right ventricle and left ventricle, the specific pressure tracings were simultaneously recorded on a PowerLab physiologic recorder System (AD Instruments, Inc., Australia). Data were not accepted if the steady state was not reached.
Metabolomic sample preparation
Blood samples from the rats were collected in 5 ml vacutainer tubes containing the chelating agent ethylenediaminetetraacetic acid (EDTA), and then, the samples were centrifuged for 15 min (1500 g, 4 °C). Each aliquot (150 µl) of the plasma sample was stored at -80 °C until ultrahigh-performance liquid chromatography equipped with quadrupole time-off light mass spectrometry (UPLC-Q-TOF/MS) analysis. The plasma samples were thawed at 4 °C, and 100 μl aliquots were mixed with 400 μl of cold methanol/acetonitrile (1:1, v/v) to remove the protein. The mixture was centrifuged for 15 min (14000 g, 4 °C). The supernatant was dried in a vacuum centrifuge. For LC-MS analysis, the samples were redissolved in 100 μl of acetonitrile/water (1:1, v/v) solvent. For monitoring the stability and repeatability of instrument analysis, quality control (QC) samples were prepared by pooling 10 μl of each sample, and these samples were analyzed together with the other samples. The QC samples were inserted regularly and analyzed every 5 samples.
LC-MS/MS analysis
Analyses were performed using an UHPLC (1290 Infinity LC, Agilent Technologies) coupled to a quadrupole time-of-flight (AB Sciex TripleTOF 6600) in Shanghai Applied Protein Technology Co., Ltd. For HILIC separation, samples were analyzed using a 2.1 mm × 100 mm Acquity UPLC BEH 1.7 µm column (Waters, Ireland). In both ESI positive and negative modes, the mobile phase contained A=25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B= acetonitrile. The gradient was 85% B for 1 min and was linearly reduced to 65% in 11 min, reduced to 40% in 0.1 min and kept for 4 min, and then increased to 85% in 0.1 min, with a 5 min re-equilibration period employed.
The ESI source conditions were set as follows: Ion Source Gas1 (Gas1) was 60, Ion Source Gas2 (Gas2) was 60, curtain gas (CUR) was 30, source temperature was 600 ℃, and IonSpray Voltage Floating (ISVF) was ± 5500 V. In MS only acquisition, the instrument was set to acquire over the m/z range 60–1000 Da, and the accumulation time for TOF MS scan was set at 0.20 s/spectra. In auto MS/MS acquisition, the instrument was set to acquire over the m/z range of 25–1000 Da, and the accumulation time for the product ion scan was set at 0.05 s/spectra. The product ion scan was acquired using information-dependent acquisition (IDA) with high sensitivity mode selected. The parameters were set as follows: the collision energy (CE) was fixed at 35 V with ± 15 eV; declustering potential (DP) was 60 V (+) and −60 V (−); excluding isotopes within 4 Da, and candidate ions to monitor per cycle: 10.
The metabolites were further confirmed by targeted metabolomic analysis or enzyme-linked immunosorbent assays (ELISAs). For targeted metabolomic analysis, multiple reaction monitoring (MRM) transitions representing the metabolites were simultaneously monitored. Each MRM ion dwell time was 3 ms, and the total cycle time was 1.263 s. ELISA was performed according to the manufacturer’s instructions.
Data processing
The raw MS data (wiff.scan files) were converted to MzXML files using ProteoWizard MSConvert before importing into freely available XCMS software. For peak picking, the following parameters were used: centWave m/z = 25 ppm, peakwidth = c (10, 60), and prefilter = c (10, 100). For peak grouping, bw = 5, mzwid = 0.025, minfrac = 0.5 were used. Collection of Algorithms of MEtabolite pRofile Annotation (CAMERA) was used for annotation of isotopes and adducts. In the extracted ion features, only the variables having more than 50% of the nonzero measurement values in at least one group were kept. Compound identification of metabolites was performed by comparing the accuracy m/z value (<25 ppm) and MS/MS spectra with an in-house database established with available authentic standards.
Statistical analysis
Statistical analysis was performed using SPSS software, version 20.0 (IBM Corp.). Continuous variables were summarized as mean ± SE and all categorical variables were expressed as proportions. Datasets containing 3 groups were first analyzed by one-way ANOVA, and significance between any two groups was further analyzed by post hoc test. For metabolomic analysis, after normalization to the total peak intensity, the processed data were uploaded before importing into SIMCA-P (version 14.1, Umetrics, Umea, Sweden), where they were subjected to multivariate data analysis, including Pareto-scaled principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). Sevenfold cross-validation and response permutation testing were used to evaluate the robustness of the model. The variable importance in the projection (VIP) value of each variable in the OPLS-DA model was calculated to indicate its contribution to the classification. Metabolites with a VIP value >1 were further applied to Student’s t-test at the univariate level to measure the significance of each metabolite. P values less than 0.05 were considered statistically significant.