Animals and experimental protocols
All the animal studies reported in this article were performed in accordance with the Guide for the Care and Use of Laboratory Animals, Eighth Edition, (2011, published by The National Academies Press). The experimental procedures were approved by Institutional Animal Care and Use Committee of Guangzhou Medical University.
C57BL/6 male mice (22–25 g body weight) were obtained from the experimental animal center of Guangdong Province (Guangzhou, China) and received a standard diet and water ad libitum. Cardiac hypertrophy was induced by permanent Transverse Aortic Constriction (TAC). Briefly, mice were anesthetized by sodium pentobarbital (50 mg/kg, ip) and artificially ventilated with an animal ventilator (DH-140, Zhejiang, China). After thoracotomy at the second intercostal space, the transverse aortic arch was ligated between the innominate artery and the left common carotid artery by the 7–0 silk suture against a 26-gauge needle. Mice underwent the same surgical procedure without TAC served as sham control. Thoracic Aorta Color Doppler ultrasound was performed 1 to 3 days after surgery to confirm the ligation of transverse aorta.
A total of 96 mice were randomized into 4 groups: (i) sham group (Sham, n = 40); (ii) TAC group (TAC, n = 40). (iii) TAC + Chloroquine (TAC + CQ, n = 8); (Ⅳ) TAC + Rapamycin (TAC + RAPA, n = 8). Following surgery, 40 mice both from the sham group and the TAC group were separated into 5 subgroups: 1W, 2W, 4W, 8W, and 12W. During a period of 1 to 12 weeks, mice in the 8W and 12W subgroup received a repeat echocardiography and/or blood pressure measurement at the indicated time point to evaluate left ventricular function and to track blood pressure. One day after each time point, mice from sham subgroup and TAC subgroup were sacrificed and the hearts were harvested and weighed to compare heart weight/body weight (HW/BW). Mice from TAC + CQ group and TAC + RAPA group received an intraperitoneal injection of CQ (10 mg/kg/day) or RAPA (1 mg/kg/day) (Sigma-Aldrich St. Louis, MO, USA) one day after TAC and delivered once daily for 8 weeks, respectively. Echocardiography was performed every two weeks to evaluate the cardiac function for the CQ-treated or RAPA-treated mice. One day after the experiment concluded, mice were sacrificed, and the hearts were harvested and processed for histologic analysis or western blotting analysis.
Blood pressure measurement
Tail-cuff method was used to measure mice blood pressure at every designated time points using a small animal non-invasive blood pressure system (Kent Scientific Corporation, USA). The heart rate (HR) and carotid SBP, DBP, and MAP were recorded by a biological signal acquisition system (CODA Monitor, USA) connected with pressure transducer.
Echocardiography
Echocardiography was performed to evaluate cardiac function by using a high-resolution imaging system (Vevo 2100, VisualSonics Inc., Ontario, Canada) equipped with a 25 MHz imaging transducer. Briefly, two-dimensional echocardiographic views of parasternal long-axis and short-axis as well as the apical four chamber were obtained. Cardiac systolic function parameters, including Left ventricular anterior wall in diastole (LVAWd), Left ventricular anterior wall in systole (LVAWs), the cardiac output (CO), left ventricle ejection fraction (LVEF), left ventricle fractional shortening (LVFS) and cardiac diastolic function parameters such as the ratio of E-wave velocity to A-wave velocity (E/A), isovolumetric relaxation time (LVRT), isovolumetric constriction time (LVCT ) and the early diastolic mitral annulus velocity/late diastolic mitral annulus velocity(e’/a’)were analyzed according to the instruction of the Vevo 2100.
Histologic analysis
The hearts were fixed in 4% paraformaldehyde, embedded by paraffin and sectioned (5 µm thickness). After routine dewaxing, the sections were stained with hematoxylin-eosin (H&E) and Masson trichrome and imaged with a microscope (Nikon Instruments Inc, Japan). By using ImageJ software (NIH, version 1.30, http://rsb.info.nih.gov/ij/), 2-dimensional cross-sectional areas of cardiomyocyte were evaluated, and the fibrosis in left ventricle (LV) was determined by the area of fibrotic tissue (blue = collagen) over LV area (above background). More than five fields in three different sections were examined for each mouse by the researcher who was blinded to the treatments.
Electron microscopy
Heart tissues were cut into sections of 1 mm3 and pre-fixed in 2.5% glutaraldehyde. After washed, the sections were incubated with 1% OsO4 at 4℃ for 3h, followed by dehydrated in graded series of ethanol, and flat embedded in epoxy resin. Ultrathin sections were counterstained with uranyl acetate and lead citrate and observed under a transmission electron microscope (HITACHI H-600, Japan).
Cell Isolation, Culture And Treatment
Sprague-Dawley pups (1-2-day-old) were purchased from the experimental animal center of Guangdong Province (Guangzhou, China). The animals were handled according to National Institute of Health Guidelines on the Care and Use of Experimental Animals. Primary cardiomyocytes were prepared as described previously.13 Briefly, ventricles were obtained from pups, and cardiomyocytes were isolated by digestion with trypsin. Cells were cultured in Dulbecco’s modified Eagles’s medium (DMEM) supplemented with 10% fetal calf serum, BrdU (10− 4 mol/L), penicillin (100 U⁄mL) and streptomycin (100 µg⁄mL). After 24 hours of serum deprivation, cardiomyocytes were treated with ET-1 (10− 7 mol/L) (Sigma-Aldrich, St. Louis, MO, USA) for 24 h to induce myocardial hypertrophy. RAPA (10− 8 mol/L), 3-methyladenine (3-MA, 5×10− 3mol/L) (Sigma-Aldrich, St. Louis, MO, USA), or CQ (5×10− 6 mol/L) was added 0.5h prior to ET-1 treatment, respectively.
Quantitation of autophagy with mRFP-GFP-LC3 adenovirus
Tandem RFP-GFP-LC3 fluorescence assay is a convenient way designed to monitor autophagic flux, by which the GFP fluorescence is quenched due to sensitive to the acidic and/or proteolytic conditions of the lysosome lumen, whereas mRFP fluorescence is not. Therefore, colocalization of GFP and mRFP fluorescence indicates an autophagosome that has not fused with a lysosome. In contrast, a mRFP signal without GFP corresponds to an autolysosome. After infected with mRFP-GFP-LC3 adenoviral particles at a MOI of 50 (Hanbio Biotechnology Co., Ltd. Shanghai, China) for 24h, the cells were treated with ET-1 for indicated time. Fluorescent signals were obtained with the confocal laser scanning microscopy (Nikon America Inc., Melville, NY). The number of autolysosomes and autophagosomes was determined by counting of red puncta or yellow puncta, respectively. Thirty randomly selected cells per experimental group were analyzed.
Immunostainning of cardiomyocytes
The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100 after treatment. After blocking with PBS containing 2% fat-free milk, the cells were immunostained by using primary antibody against TFEB (1:200, Cat#BS8832, Bioworld Technology, St. Louis Park, MN, USA) and the second antibody conjugated to Alex-488 (green) (1:1000, Santa Cruz, CA, USA). F-actin of myocytes was probed using phallodin conjugated to TRITC (red) (Sigma-Aldrich, St. Louis, MO, USA) and nuclei were co-stained with DAPI (blue) (Invitrogen, Grand Island, NY). Fluorescent signals were obtained with a fluorescence microscope (Olympus 1×2-UCB-2).
Cell size measurement
F-actin of cardiomyocytes was immunostained using phallodin conjugated to TRITC. Myocyte size was assessed by immunofluorescent microscopy and measured by ImageJ software. Thirty randomly selected cells per experimental group were analyzed.
Diacylglycerol (DAG) assay
Cell lysates were prepared in RIPA buffer and clarified by centrifugation. DAG levels were measured using enzyme-linked immunosorbent assay (ELISA) kits (Cloud-Clone Corp, Houston, TX) following the manufacturer’s instructions and DAG concentration in each sample was calculated based on the standard curve by a microplate reader (BioTek Synergy2; BioTek, Winooski, VT, USA).
Generation of TFEB shRNA construct
shRNA against TFEB was constructed into pLKO.1 lentiviral vector (Open Biosystems, Ottawa, Canada) following the manufacturer’s instruction. The construct was verified by DNA sequence analysis.
Lentiviral Preparation And Infection
Lentivirus expressing shRNAs against DGKζ or TFEB were prepared by co-transfecting DGKζ shRNA lentiviral plasmids (TRCN0000025394, TRCN0000025395, TRCN0000025398) (Open Biosystems, Ottawa, Canada) or TFEB shRNA lentiviral plasmids with packaging plasmid into HEK-293T cells using FuGENE6 reagent (Roche, Indianapolis, IN, USA). Empty lentiviral plasmid (pLKO.1) and lentiviral plasmid containing scramble sequences served as vector control and non-silencing control, respectively. Cardiomyocytes were infected by adding 50 MOI lentivirus particles to the medium for 24 hours, and then cultured in the newly replaced medium for another 24 hours before serum deprivation and ET-1 treatment. Specificity and degree of knockdown were confirmed by western blotting 72 h after virus infection.
Co-immunoprecipitation (IP) and western blotting analysis
Cell lysates were prepared in RIPA buffer and TFEB precipitates were generated from cardiomyocyte protein extracts with anti-TFEB antibody and protein A/G beads (Santa Cruz, CA, USA). Immunoprecipitates and the proteins extracted from heart tissues or cardiomyocytes were separated by SDS-PAGE and transferred to PVDF membranes (Roche Molecular Biochemicals, Mannheim, Germany). The membranes were blocked and detected with anti-LC3 antibody (Cat#3868), anti-Beclin-1 antibody (Cat#3738), anti-p62 antibody (Cat#5114), anti-phosphorylated-Akt antibody (Cat#9271), anti-Akt antibody (Cat#9272), anti-phosphorylated-AMPK antibody (Cat#2531), anti-AMPK antibody (Cat#2532), anti-phosphorylated-mTOR antibody (Cat#2971), anti-mTOR antibody (Cat#2972) from Cell signaling technology (Beverly, MA), anti-TFEB antibody, anti-DGKζ antibody (Cat#SC-8722, Santa Cruz, CA, USA) or anti-β-actin antibody (Cat#BS6007M, Bioworld Technology, St. Louis Park, MN, USA), respectively. The density of target bands was accurately determined by the computer-aided Quantity One analysis system. To avoid variability from different treatment time, mice or the proteins for detection per se, the expression of proteins in the heart of TAC mice was normalized to that of the sham mice in each time point, and β-actin served as a loading control.
Hypertrophic gene expression
The expression of fetal genes of brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) was assessed by RT-PCR. Briefly, total RNA was extracted by TRIzol reagent (TaKaRa Biomedical Technology, Beijing, Co., Ltd), and equal amount of purified RNA was reversely transcripted using the RNA PCR Kit according to the manufacturer’s instructions. The cDNAs were amplified and real-time PCR was performed with primers specific for BNP and β-MHC (Life technology, Invitrogen, Ltd. Paisley PA4 9RF, UK). The levels of hypertrophic gene mRNAs were normalized to that of β-actin mRNAs.
Statistical analysis
All data were expressed as mean ± standard deviation and analyzed by software of GraphPad Prism 5.0 (GraphPad Software Inc). Student t test was used to evaluate the differences between two groups, while one-way ANOVA was used for more than two groups followed by Tukey post-hoc test. A value of P < 0.05 was considered as statistically significant.