All procedures involving animals were approved by Harbin Medical University Ethics Committee for Animal Experiments and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
Pig model of pressure-overloaded left ventricular hypertrophy
Ten 8- to 10-week-old Bama Miniature Pigs weighing 10 to 15 kg were sedated with an intramuscular injection of diazepam (0.4 mg/kg body weight) and ketamine (20 mg/kg body weight). Once anesthetized, piglets were intubated and mechanically ventilated. Intravenous injection of 3 % pentobarbital sodium was given to maintain anesthesia throughout the operation. To start, a left lateral thoracotomy was performed in the third intercostal space. The pericardium was incised, and care was taken to avoid damage to the phrenic nerve. A piece of suture inside a silicone tube was then placed to circle the ascending aorta. Followed by that the ends of the suture were tied to allow for overlap of the tube ends to create a peak systolic pressure gradient of 10 to 20 mm Hg between the left ventricle (LV) and aorta distal to the stenosis. The operation finished by closing the chests and the animals were allowed to recover for 12 weeks for the development of LV hypertrophy.
The protection model of hypertrophy myocardium
Twelve weeks after aortic banding, the chests were reopened under a general anesthesia. Firstly the pericardium was opened longitudinally along the midline. After animal heparinization (3,000 IU heparin into a peripheral vein), the aorta, pulmonary artery, and inferior and superior vena cava were dissected and clamped. Conventional cardiopulmonary bypass was established with ascending aortic and single right atrial cannulation with systemic normothermia (36.5 °C-37 °C), perfusion flow rate 250 ml/min and retrograde perfusion pressure at 80-120 mmHg. An aspirator was placed near the coronary sinus through an incision under the right atrium. The effluent was collected continuously by the aspirator.
Pig blood was collected from the animal chest and mixed with Krebs-Henseleit solution in a 1:1 ratio for perfusion. Krebs-Henseleit solution is widely accepted as a physiologic perfusion medium and has been used for many years for heart perfusion. The Krebs-Henseleit solution contained 118 mmol/L NaCl, 1.2 mmol/L MgSO4, 0.5 mmol/L ethylenediaminetetraacetic acid, 11 mmol/L glucose, 25 mmol/L NaHCO3, 1.75 mmol/L CaCl2 and 0.625 % bovine serum albumin. The concentration of potassium was 4.0 and 16 mmol/L for normothermic normokalemic simultaneous perfusion (NNSP) and normothermic hyperkalemic simultaneous perfusion (NHSP), respectively. The temperature of the heart was maintained at 36.5 °C to 37 °C throughout the operation.
Ten hypertrophied hearts were divided into two groups with five hearts in each group. Group 1 (n = 5) were used to evaluate the effect of empty beating (NNSP). Group 2 (n = 5) were used to evaluate the effect of cardioplegic arrest (NHSP). Both groups underwent a protocol consisting of 80 min of either NNSP (group 1) or NHSP (group 2). The 80-min preservation period was chosen because it was sufficient for most valve surgeries. Intermittent delivery was used for normothermic cardioplegic arrest in this study. After the 80-min preservation period, the aortic cross clamp was removed in both groups. Normal perfusion of was given and patients core temperature stayed 35–37 °C. The sternum was subsequently closed.
Cardiac contractive function and LV wall thickness were assessed using cine MRI immediately after the surgery. The total time interval was less than 20 min. Myocardial energy metabolism was monitored by using phosphorus 31 magnetic resonance spectroscopy. MR perfusion imaging was used to evaluate myocardial perfusion. All of the MRI images were collected by Philips Achieva 3.0 T superconducting MRI scanner.
After imaging examination, the animals were killed, autopsied and the myocardial specimens were harvested. Both the heart and LV weight (LV free wall and septum) were measured. Corresponding data of normal pig hearts, used as control for this study, was referred to another study of us which had not been published yet (data unpublished). Tissues of LV (with coronary artery) were taken for lab experiment. Western blot analysis was carried out to determine the expression of Troponin I (cTnI), Troponin T (cTnT), smooth muscle myosin heavy chain (SM-MHC), PARP4 and Casapase-3. TUNEL assay was used to detect apoptotic cardiomyocytes.
Assessment of contractile function
Left ventricular end-systolic volume and left ventricular end-diastolic volume were measured by the cine MRI. Contractile ability of the hypertrophied hearts was also assessed by left ventricular ejection fraction [(left ventricular end-diastolic volume and left ventricular end systolic volume) / left ventricular end-diastolic volume].
Phosphorus 31 MR spectroscopy
Phosphorus 31 (31P) MR spectroscopy was performed on a 3-T magnet equipped. The 31P MR spectra were acquired using a MR surface coil. Monitoring 31P MR signal intensity was performed using a point resolved spectral selection (PRESS) sequence during the test. Phosphorus compounds included phosphocreatine (PCr), inorganic phosphate (Pi) and 3 peaks (α, β and γ) of adenosine triphosphate (ATP) were observed and noted. The β peak was used for quantifying ATP.
MR perfusion imaging
The blood perfusion of hypertrophy myocardial tissue was evaluated by MR perfusion imaging. Monitoring MR perfusion imaging signal intensity was performed using a echo-planer imaging (EPI) sequence. The pig hearts in groups 1 (n = 5) and 2 (n = 5) were subjected to the injection of Gd-DTPA (0.05 mmol/kg body weight; Magnevist, Berlex, Canada). 10 min after contrast agent was injected, late enhancement was performed using a phase sensitive inversion recovery (PSIR) sequence.
Freshly frozen myocardial tissue samples were homogenized in RIPA buffer. Protein from myocardium tissues was extracted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein
s was then transferred to nitrocellulose membranes, blocked and probed sequentially with primary antibodies against cTnI, cTnT, SM-MHC, PARP4 (Bioss Biotechnology, Inc) and Casapase-3 (Cell Signaling Technology, Inc). After incubation in the primary antibody, the membrane was incubated in an appropriate secondary antibody. After washing, the bound antibody complexes were detected using an electrochemiluminescence reagent.
Apoptosis detection and quantification by TUNEL assay
Detection of apoptosis was carried out by a terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assay (Roche Diagnostics GmbH). Approximately 20 randomly chosen, non-overlapping images covering the infarct and border regions were acquired using a × 200 objective. Blinded observers counted the number of TUNEL positive nuclei in each image. The result was then expressed as the number of TUNEL positive nuclei per mm2 area.
All the data collected from MRI were analyzed by using the cardiac analysis package in the post-processing workstation. All numerical results were expressed as the mean ± S.D. of the mean. Student’s t test (unpaired) was used to analyze the differences between NHSP and NNSP. A value of P less than 0.05 indicates significant difference.