Preparation of shRNA and microbubble(MB)
According to the manufacturer’s instructions, the SonoVue (Bracco Medical Technology Co.,Ltd.Shanghai,China) were dissolved in 5ml 0.9% saline solution, which was thoroughly mixed with MBs and vibrated until the MBs were well distributed. The resulting concentration of MBs was 2.5μm and the density was (1.0-5.0)*108/ml.
Following Bax-ShRNA plasmid labeled with green fluorescent protein marker (5μl/well,Invitrogen,U.S.) and polyethyleneimine(PEI) (3.75μl/well,Sigma Aldrich,U.S.) were mixed, serum-free DMEM(91.25μl,Hyclone,U.S.) was added in for incubation at room temperature for 10min. MBs were mixed with the Bax-ShRNA/PEI in a 1:1 volume ratio till analysis. Meanwhile, the mixture of MBs and Bax-ShRNA was used as a control. Negative control transfection was performed using Lipofectamine 2000 as instructed.
Cell lines and cultures
Rat cardiomyocyte H9c2 purchased from Cell bank of Chinese Academy of Sciences, was cultured in DMEM supplemented with 10% fetal bovine serum(Hyclone,U.S.), 1% penicillin and streptomycin. Slides of 21mm diameter were used as H9c2 adhesion substrate. The 0.25ml polylysine diluted by phosphate buffer saline(PBS) solution was dropped onto the sterile slides, which were placed overnight at 4℃. The polylysine slides were washed twice with sterile water the next day, and then set into the bottom of 6-well plates.
H9c2 cells were seeded onto the 6-well plates at a density of 5*105 cells/well and split into 5 group before transfection: (1) Bax-ShRNA;(2) Lipofectamine 2000+Bax-ShRNA;(3)UTMD+Bax-ShRNA;(4)UTMD+Bax-shRNA/PEI;(5)Bax-shRNA/PEI +MBs without ultrasound exposure(US).
Cell transfection
H9c2 reaching a 70% cell confluence in the 6-well plates were washed with saline solution and serum-free medium respectively, and then added in serum-free DMEM 1ml/well. US was treated (1 MHz, 20% duty cycle, 1 W/cm2, pulse duration 60s) using a non-imaging ultrasound probe (Low power focused ultrasound experimental device; Chattanooga company, USA). Six hours later, serum-free DMEM was changed into DMEM with 10% serum and the H9c2 were continued to be cultured in an incubator for 48 hours.
Bax knockdown with Bax-shRNA in vitro
At 48h after treatment, the infection efficiency was assessed by green fluorescent protein expression of H9c2 cells in different groups using fluorescence microscopy. The transfected H9c2 were washed with PBS and analyzed for the expression levels of Bax-mRNA by real-time PCR (RT-PCR) and western blotting[15].
Total tissue RNA was extracted by TRIzol (Ambion, US) method. Primer synthesis was completed by Invitrogen Technologies (Shanghai, China). Primer sequences for Bax were as follows: upstream: 5’-GGATGCGTCCACCAAGAA-3’ and downstream:5’-TCCCGGAGGAAGTCCATT-3’.
The Bax protein in each group was extracted by RIPA lysis buffer, quantified by the BCA kit (P0012, Beyotime, China), and kept at -80℃ for subsequent applications. The protein was separated and then sealed using PBST at room temperature for 2h after membrane transfer. The membrane was probed with mouse monoclonal antibodies overnight at 4℃. After using PBST to wash membrane 3 times, the membrane was reprobed with secondary mouse anti-rabbit IgG for half an hour at room temperature. Western blots were rinsed with PBST buffer for 3 times and reacted with ECL solutions.
Heart transplantation models
30 healthy male Wistar rats, body weight(266±12g), 7-10 weeks old and the other 30 healthy male Sprague Dawley (SD) rats, body weight(281±14g), 7-10 weeks old were all purchased from Shanghai SLAC Laboratory Animal Ltd, and raised in specific pathogen-free animal center according to the standard animal management guidelines. The hearts of SD rats were transplanted to Wistar rats serving as the allograft group. The animal experiment was approved by the Tongji University Institutional Animal Care and Use Committee.
Heterotopic cardiac transplantation was performed according to the Ono-Lindsey method[16, 17]. Rats were anesthetized with isoflurane (2%) and ventilated using a rodent ventilator (Harvard Apparatus Inc.) throughout the surgical procedure.After being anesthetized, middle incision thoracotomy of donor rat was performed, then the heart was taken out and the ascending aorta and pulmonary artery were dissected in cold saline. The abdomen of recipient rat was opened by a midline incision, and the abdominal aorta and the postcava were isolated and occluded. The aorta and pulmonary artery of donor rat were anastomosed end to side to the abdominal aorta and the postcava of the recipient rat, respectively. After heart reperfusion, the abdomen was closed and the rat was placed in an incubator to recover. The rats were euthanized with an overdose of sodium pentobarbital (P010, Merck, Germany, 250 mg/kg). All methods were carried out in accordance with the Institutional Animal Care and Use of Laboratory Animals.
Targeted therapy of Bax-shRNA in vivo
Based on in vitro transfection results, three groups were created (n=10 rats in each group):Group A, Allograft (ALLO); Group B, ALLO+cyclosporine(CsA,NOVARTIS Pharma Ltd). CsA were given at a dose of 5 mg/kg per day until 6 days after transplantation; Group C, ALLO+UTMD+Bax-shRNA/PEI (200μL MBs mixed with 30μg Bax-shRNA and 22.5μL PEI). UTMD was performed 30s after injection of Bax-shRNA/PEI via the aorta using the same therapeutic ultrasound probe in vitro and the following irradiation parameters: frequency (1 MHz), duration(3 min), output intensity (2.0W/cm2), duty cycle(20%).
Western blot and histopathological analysis
At 6 days post transplantation, 5 recipient rats of each group were sacrificed and the hearts were removed. Left ventricular myocardium tissue were paraffin-embedded and made into 5μm slices. The expression of green fluorescent protein in myocardium was observed by fluorescence microscopy. The procedure of western blotting was the same as experiment procedure in vitro. The other 5 rats in each group were observed for the survival time of heterotopic cardiac allograft.
Histopathological analysis was performed with hematoxylin-eosin (H&E) staining. The rats were graded according to the standards for acute heart allograft rejection established by International Society for Heart and Lung Transplantation (ISHLT) in 1990 [18].
Statistical analysis
The measurement data was expressed as mean ± SD and analyzed using SPSS 21.0 software. Statistical analyses were either performed with Student’s t-test or by one-way analysis of variance(ANOVA). Significant differences were considered at p value < 0.05.