Cell Lines and Culture Conditions
AC16 human adult ventricular cardiomyocyte cells were purchased from the American Type Cell Culture (ATCC). The cells were maintained in Dulbecco’s Modified Eagle’s (DMEM)/F-12 medium (Gibco Invitrogen), supplemented with 12.5% (v/v) fetal bovine serum (Gibco Invitrogen) and 1% (v/v) penicillin/streptomycin (Gibco Invitrogen) at 37 °C in a humidified incubator containing 95% O2 and 5% CO2. The media was refreshed every 3 days. Cells cultured to approximately 80% confluence were treated with PE (100 mM, Sigma) alone under serum-free conditions, and both rapamycin (100 nM, Sigma), or AZD2014 (range from 10 to 100 nM, dissolved in distilled water, Selleck) under serum conditions with daily treatment.
Western Blot Analysis
Cells were washed with Dulbecco's Phosphate-Buffered Saline (DPBS, Gibco Invitrogen) lysed with 200 μL of RIPA buffer, and centrifuged at 13,000 rpm for 20 min. Supernatant was collected in 1.5 mL tubes. Protein concentration was determined using a BCA assay kit (Life Technologies). 20 μg of total protein of samples was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and then these proteins were transferred to PVDF membranes (Millipore). Protein transferred membranes were blocked with 5% (v/v) of skim milk in TBS containing 0.1% Tween-20 (TBS-T) for 30 min. Membranes were washed three times with TBS-T and incubated with primary antibody in 5% (v/v) skim milk in TBS-T at 4 °C for overnight. After overnight incubation, membranes were washed three times with TBS-T and incubated with secondary antibody in 5% skim milk in TBS-T at RT for 1 h. Immunoreactivity was detected using Clarity™ western ECL substrate (Bio-Rad) and by G:BOX Chemi XX6/XX9 gel doc systems (Syngene, Cambridge, UK). The The total-Akt (t-Akt), phospho-Akts473 (p-Akts473), total-mTOR (t-mTOR), phospho-mTORs2448 (p-mTORs2448), total-S6 (t-S6), phospho-S6s240+S244 (p-S6s240+S244) antibodies were obtained from Abcam. The horseradish peroxidase-conjugated anti-mouse IgG secondary antibody and anti-rabbit IgG were obtained from Abcam. Signals of t-Akt, p-Akts473, t-mTOR, p-mTORs2448, t-S6 and p-S6s240+S244 were quantitatively analyzed using NIH ImageJ software.
RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. mRNA was reverse-transcribed into complementary DNA (cDNA) using iScript™ cDNA synthesis kit (Bio-Rad). The quantitative PCR analysis was performed by iTaq Universal SYBR Green Supermix (Bio-Rad) with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) according to the instructions. Target gene expression was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for quantification. The resulting templates were subjected to PCR using the following specific primers: GAPDH (sense 5’-ACCCACTCCTCCACCTTTGAC-3’, antisense 5’-TGTTGCTGTAGCCAAATTCGTT-3’); ANP (atrial natriuretic peptide, sense 5’-GACAGACTGCAAGAGGCTCC-3’, antisense 5’-GCTGCAGCTTAGATGGGATGA-3’), and BNP (B-type naturetic peptide, sense 5’-TCAGCCTCGGACTTGGAAAC-3’, antisense 5’-CTTCCAGACACCTGTGGGAC-3’).
FACS Analysis
Cell surface antigens on cells were evaluated by fluorescence activated cell sorting (FACS) analysis. For FACS, cells were dissociated by 0.05% trypsin/EDTA (Highclone), washed with PBS, fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with a mixture of bovine serum albumin (BSA). Next, samples were stained with antibodies against Anti-Myh6 Actin (alpha myosin heavy chain actin, Abcam, 1:200) and Anti-ANP antibody (R&D, 1:200) for 30 min or 1 h at 4 °C. Also, Anti-Goat IgG was used for isotype control. Samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor-488 conjugated goat anti-mouse (Abcam, 1:400) and Alexa Fluor-647 conjugated donkey anti-goat (Abcam, 1:400)) for 30 min at 4 °C. Corresponding mouse/rabbit isotype antibodies were used as controls. Cell immunotypes were determined by BD FACS CantoII (BD Biosciences) and the percentage of expressed cell surface antigen was calculated for 10,000 gated-cell events.
Immunostaining
Samples were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with a mixture of BSA. Samples were incubated with primary antibodies (Anti-Myh6 Actin (Abcam, 1:200) and Anti-ANP antibody (R&D, 1:200) overnight at 4 °C and washed. Samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor-488 conjugated goat anti-mouse (Abcam, 1:400), Alexa Fluor-647 conjugated donkey anti-goat (Abcam, 1:400)) for 30 min at at RT and 4′,6-diamidino- 2-phenylindole dihydrochloride (DAPI) as a nuclear counterstain for 5 min at R.T. Samples were washed and mounted for imaging on a fluorescence microscope (Leica DMI-6000, Leica). The relative surface area of coverage for stains was quantified with ImageJ software (NIH).
Administration of AZD2014
Myosin-binding protein C (Mybpc3)-targeted knockout (KO) mice recapitulate typical aspects of human hypertrophic cardiomyopathy. Mybpc3-targeted ablation in the KO mice is sufficient to trigger profound cardiac hypertrophy, impaired systolic and diastolic function, and depressed myocyte contractile properties [22]. All experiments were approved by the University of Arizona Institutional Animal Care and Use Committee and followed the U.S. National Institutes of Health Using Animals in Intramural Research guidelines for animal use. Seven-week-old Mybpc3 KO mice (male) were used to examine the effect of AZD2014. The mice were given AZD2014 at doses of 2.5 mg or 10 mg/kg on IP injection twice a week for 12 weeks.
Echocardiography
Survival was monitored and echocardiography was performed at 12 weeks, at the end of the course of treatment. All imaging studies were performed using the VisualSonicsVevo® 770 imaging system (Scanhead: RMV707B, 15–45 MHz). Anesthesia was induced for one minute in an induction chamber (3% isoflurane mixed with 97% O2 at a flow rate of 1 L/min). After placing the mouse in a supine position atop a pad with embedded ECG electrodes, anesthesia was maintained via inhalation of 1.5–2% isoflurane and 98–98.5% O2 at a flow rate of 1 L/min using a nose mask. The ECG signal was monitored throughout the procedure. After immobilizing the mouse on the echocardiography stage with tape, chest hair was removed with hair removal cream and a layer of preheated ultrasound gel was applied to the chest. Body temperature was monitored throughout the whole procedure by an inserted rectal probe and maintained within a narrow range (37.0 °C ± 1.5 °C) via the heated platform and a heat lamp. Standard measurements of were performed in systole and diastole in parasternal long-axis projection and the functional parameters were calculated from M-mode measurements.
Histology
At the endpoint of each experiment, mice were anaesthetized with 4% isoflurane and subsequently euthanized by cervical dislocation. The heart was harvested, washed, and fixed for histological analyses. Cross-sections of the hearts made along the transverse midventricular tissue were stained for hematoxylin and eosin (H&E).
Statistical analysis
Data are expressed as means ± standard deviation (SD) from at least three independent experiments and were analyzed using one-way ANOVA and Tukey’s post-hoc test. In all cases, p < 0.05 was considered to be statistically significant.