Animals
This study was performed in adherence with the National Institutes of Health Guidelines for the Use of Laboratory Animals, and was approved by the Ethical Review Committee of Experimental Animal Welfare of Nanjing Medical University (IACUC-1712011). Thirty male Sprague‑Dawley rats weighing 230±10g were obtained and twenty-four rats were given a single subcutaneous injection of SU5416 (20mg/kg, MCE, HY-10374). Then, they were placed into normobaric hypoxia chambers (CYES‑II; Shanghai Anting Scientific Instrument Factory) with 10±1% oxygen. The CHRs were divided into four groups (1w/2w/3w/5w, n=6 respectively). The remaining six rats were raised as the control group.
31P magnetic resonance spectroscopy
All the MR procedures were performed weekly in 1,2,3,5weeks of modeling separately on a 7.0 Tesla horizontal magnet (Bruker BioSpec 7T). Animals were anesthetized using gaseous isoflurane in oxygen (3% box induction), and maintained at 0.5-2.5% during the entire MR procedures.
In this study, we took a method called “outer volume suppression” as a reference and optimized it [10]. A 20mm diameter dual-tuned (31P/1H) surface coil was used for transmission and reception. A standard non localized pulse-acquisition sequence with 6 spatially selective saturation bands surrounding the volume of interest were used to avoid the signal contamination from the structures around the heart. Acquisition parameters were: 50 μsec block pulse, 23°flip angle, 1.5 s repetition time, 10kHz band width (BW), 128 averages.
Cardiac energy status was evaluated by means of PCr/ATP ratio calculated by dividing PCr with γ-ATP resonances using the spectral analysis software (TopSpin versions 4.0 )(Fig. 1).
Magnetic resonance imaging for ventricular size and function
The system was equipped with a 72-mm inner diameter volume coil. The heart of was placed at the center of the coil. Prospective-ECG and respiratory-gated cine images were acquired for getting four-chamber and short-axis views of heart, with the following parameters: TE=2.5ms, TR=8ms, flip angle=15°, field of view=4.7*5.0cm, 192×192 matrix, in-plane resolution=244 μm, slice thickness=1.5mm, and 14-23 frames/heart beat. A set of 5 short-axis images was used to cover the heart.
Left and right ventricular volumetric parameters were obtained using the image analysis software (Circle CVI 42, Circle Cardiovascular Imaging Inc.) by outlining the end myocardium of both ventricles during end-systolic and end-diastolic phases. The volumes of ventricles were indexed to body surface area (BSA), which is calculated by the formula of BSA(m2)=0.91*weight(g)2/3/1000. The right ventricular ejection fraction (RVEF), right ventricular end-diastolic volume index (RVEDVi), right ventricular end-systolic volume index (RVESVi), left ventricular ejection fraction(LVEF), left ventricular end-diastolic volume index (LVEDVi), and left ventricular end-systolic volume index(LVESVi) were acquired.
Hemodynamic measurements
Every rat was performed with right heart catheter after MR procedures. Rats were weighed and anesthetized with 1.0 g/kg urethane (Sigma‑Aldrich), administered intraperitoneally. A polyethylene catheter (PE10, 427400; BD Biosciences,) and heparinsaline (125 U/ml; Changzhou Yinsheng Pharmaceutical Co., Ltd.) was inserted into the right jugular vein and advanced into the right ventricle. The catheter was connected to an MPA Acquisition and Analysis system (MP100; BIOPAC Systems, Inc.) by a pressure transducer (TSD104A; BIOPAC Systems Inc.). The right ventricular systolic pressure (RVSP) was recorded using a multiparameter monitor PM‑8000 (Zhuhai Joyful Medical Equipment Co.) [11].
Following measurements of hemodynamic parameters and blood sample collection, the rats were sacrificed by cervical dislocation, and the thorax was opened. The heart was removed and the right ventricle (RV), left ventricle (LV) were separated. The mass ratio of RV to LV (RV/LV) was evaluated.
Histological analysis
The cardiomyocyte hypertrophy was observed based on hematoxylin and eosin (H&E) staining. The myocardial fibrosis was assessed using Masson’s trichrome staining. Three 1mm3 pieces were taken from the RV, LV and S separately for transmission electron microscope (TEM) observation. Myocardial mitochondria was observed under the JEOL-1010 TEM.
Statistical analysis
Experimental measurement data was expressed as Mean ± SD () and was statistically processed by SPSS 24.0 software (IBM Corporation) using one-Way ANOVA analysis followed by paired or unpaired t-test. Pearson correlation analysis was used for correlation analyses. Results were considered significant at p<0.05.