Animals and animal mode
C57BL6J were came from the and raised at the SPF Laboratory Animal Center of Nanjing Medical University. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (Nanjing, China).
For mice aortic banding (AB) model, mice (aged 8-10 week, 23.5-27.5mg) were subjected to aortic banding as previous study described. Briefly, mice were anesthetized with sodium pentobarbital, and the mice were placed in the left lateral decubitus position after skin preparation. The mouse skin was cut, and the mouse aorta was exposed at the 3rd to 4th costal margins. The aorta was ligated with a 27G needle. After the ligation was successful, the needle was removed. The mice in the sham operation group were only hung up without ligation. The mouse skin was sutured layer by layer, and analgesics were administered three days after the operation. To knockdown TMEM43, mice were injected with adeno-associated virus 9 (AAV9)-shTMEM43 (from Vigene Bio-tek, Shanghai, China) or the control AAV9 (ScRNA) one week before AB surgery.
Adeno-Associated Virus Vector
Recombinant AAV9-shTMEM43 and the control AAV9-scRNA were constructed by Vigene Bioscience Company (Shanghai, China) with a short, small Troponin T (TnT) promoter was used to induce cardiomyocytes specific gene delivery. A total of 60–80 ml of AAV9-shTMEM43 or AAV9-scRNA (5.0–6.5 × 1013 VG/ml) was injected into the mice via tail vein 1 week before AB surgery.
Transthoracic echocardiography was performed as previously described[13, 14]. Isoflurane (1.5%) was used to anaesthetize the mice, and echocardiography was performed with a 10-MHz linear-array ultrasound transducer to obtain M-mode echocardiography data. The left ventricle (LV) end-diastolic dimension (LVIDd) and LV end-systolic dimension (LVISd) were obtained, and the LV ejection fraction (LVEF) and LV fractional shortening (LVFS) values were calculated. A total of 10 mice from each group were subjected to transthoracic echocardiography.
Hematoxylin & eosin (HE), PSR staining
Hematoxylin & eosin (HE) staining was used to valuate cross section area as previous described. Image-Pro Plus 6.0 was used to analyze 10 sections from each heart and 6 hearts from each group. PSR staining was used to show collagen volume. For the fibrosis area calculation, Image-Pro Plus 6.0 was used to analyze 6 sections from each heart and 6 hearts from each group.
Cardiomyocyte isolation and culture
Neonatal rat cardiomyocyte (NRCM) culture was performed as previously described[13, 14]. Briefly, the hearts of Sprague-Dawley rats (1-3 days old) were quickly removed, and ventricles were preserved and digested with 0.125% trypsin-EDTA (Gibco) 4 times for 15 min each time. Digestion was halted with DMEM-F12 supplemented with 15% fetal bovine serum (FBS, Gibco, USA). After 5 digestion reactions, the cells were collected and incubated in a 100-mm dish with DMEM-F12 supplemented with 15% FBS. After 90 minutes, the cell culture medium was collected, and NRCMs in the upper layer of the cell medium were removed and seeded onto a 6-well plate to exclude the non-cardiac myocytes adhered to the bottom of the 100-mm dish. NRCMs were identified by α-actin staining.
Cells were transfected with adenovirus (Ad-) to overexpress TMEM43 (Ad-TMEM43, MOI = 50, Vigene Bioscience, Jinan China). Then, the cells were stimulated with 50 μM phenylephrine (PE) for 48h to induce cardiomyocytes hypertrophy response.
Western blot and qPCR
Total protein was isolated from heart tissues then subjected to SDS-PAGE (50 μg per sample). After transfer onto Immobilon membranes (Millipore, Billerica, MA, USA), proteins were incubated overnight at 4°C with primary antibodies against totel (T) NF-κB and phosphorylated (P-) NF-κB purchased from Abcam (1:1000 dilution), and GAPDH (1:1000 dilution) purchased from (Cell Signaling Technology (1:1000 dilution). Blots were developed with enhanced chemiluminescence (ECL) reagents (Bio-Rad, Hercules, CA, USA) and captured by a ChemiDoc MP Imaging System (Bio-Rad). GAPDH served as an internal reference protein.
Total RNA (2 μg per sample) from frozen mouse heart tissue and cardiomyocytes was reverse transcribed into cDNA using the oligonucleotide (DT) primer and the transcript first strand cDNA synthesis kit (Roche). Then, a light Cycler 480 instrument (software version 1.5, Roche) and the SYBR green PCR master mix (Roche) was used to perform RT-PCR. All genes were normalized using GAPDH.
The cells were seeded on the cell slides, and the treated cells were fixed with 4% formalin, permeabilized with 0.2% Triton X-100, blocked with 8% goat serum, and then given the corresponding primary antibody, such as a-actin or P-NF-κB, purchased from Abcam (1:100 dilution). Cells were subsequently enriched with fluorescent secondary antibodies, and nuclei were stained with DAPI. Then photographed with a fluorescence microscope (Olympus DX51, Tokyo, Japan).
All data are expressed as the mean ± SD. Differences among groups were analyzed by two-way analysis of variance followed by Tukey’s post hoc test. Comparisons between two groups were analyzed by an unpaired Student’s t-test. P values less than 0.05 indicated statistical significance.