Cell culture and treatment
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were available from Beijing Cellapy Biotechnology Co., Ltd. (Beijing, China), and the rat heart-derived H9c2 cardiomyocytes were available from the Chinese Academy of Sciences (Shanghai, China). Both cardiomyocytes were grown in DMEM (Dulbecco’s Modified Eagle Medium; Invitrogen, Carlsbad, CA, USA) under 5% CO2 at 37 °C. In all, 10% FBS (fetal bovine serum; HyClone, South Logan, UT, USA) and 1% antibiotics acted as the medium supplements. The in vitro CH cell model was established in both cardiomyocytes with the 24 h of treatment of 150 nM of Ang II (Sigma-Aldrich, St. Louis, MO, USA) or 50 μM of isoproterenol (ISO; GuideChem, China) for 24 h. In total, 3 U/μg of RNase R was procured from Epicentre Technologies (Madison, WI, USA). The cycloheximide (CHX; 40 μg/mL) was also acquired from Sigma-Aldrich (St. Louis, MO, USA). The 3-methyladenine (3-MA; an autophagy inhibitor) was obtained from Abcam (Cambridge, MA, USA).
Plasmid transfection
The designed shRNAs and NC-shRNAs (Genepharma Company, Shanghai, China) were applied for silencing circ-SIRT1 (or circ-Sirt1), USP22, and SIRT1. Besides, miR-3681-3p mimics/inhibitor and NC mimics/inhibitor as well as the pcDNA3.1(+)/circ-SIRT1 (circ-SIRT1-oe) or pcDNA3.1(+)/circ-Sirt1 (circ-Sirt1-oe) or empty pcDNA3.1(+) CircRNA Mini Vector (pcDNA3.1(+)), were all procured from Genepharma Company. All plasmids were transfected into hiPSC-CMs and H9c2 cardiomyocytes for 48 h employing the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates.
Animal study
To establish the CH mouse model, male C57BL/6 mice (n = 12), aging 8-week-old and weighing 20–25 g, were commercially acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animal-related protocol was approved by the Animal Care and Use Committee of the First Hospital of China Medical University. The CH mouse model was established via Transverse Aortic Constriction (TAC) surgery (N = 6). After anesthesia with intraperitoneal ketamine and xylazine, the transverse thoracic aorta was dissected from the mouse model. Mice were then placed on a ventilator for 1 week to recover the mouse model. The pressure gradient in the TAC group was examined by echocardiography. Mice of the sham group (N = 6) were subjected to the same experimental procedures as the TAC group, except the aorta seam. Besides, we constructed Ang II-treated in vivo model by chronic subcutaneous infusion of Ang II into mice (N = 6), saline as control (N = 6). To further analyze the effects of circ-Sirt1 on Ang II-treated in vivo model, adenovirus (Biocobio, Tianjin, China) of circ-Sirt1 or pcDNA3.1(+) was injected into the mice left ventricle myocardium 7 days before TAC surgery.
Enzyme-linked immunosorbent assay (ELISA)
Serum was diluted using PBS, cardiac tissues with the same weight in the sham and TAC groups were prepared via homogenization in PBS. Ang II concentrations in serum were measured via ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) following the supplier’s protocols. All samples were analyzed via two bio-repeats and two technical replicates.
Immunofluorescence staining for cell surface area measurement
Mice heart cells, cardiomyocytes (hiPSC-CMs and H9c2) were seeded in 96-well plates and then washed twice with phosphate-buffered saline (PBS). Subsequently, cardiomyocytes were fixed by 4% formaldehyde for 20 min, permeated by 0.1% Triton X-100, and then incubated with α-actinin antibody (Cat #: 6487; 1/100, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. The secondary antibody (Cat #: 4410; 1/1000, Cell Signaling Technology) was used to culture cells at room temperature for 1 h. After DAPI staining, immunofluorescence was observed under a fluorescence microscope (Olympus Corp., Tokyo, Japan) for the estimation of cell surface area. In all, 50 independent cells were analyzed per sample. The researchers performing the analysis were blinded to the experimental groups. Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates. The results were analyzed with Image-Pro Plus Data Analysis software.
RNA extraction and RT-qPCR
TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) was used for the extraction of total RNA from cardiomyocytes. Then, the reverse transcription was accomplished as per the supplier’s instructions (Invitrogen, Carlsbad, CA, USA). To examine gene expression, RT-qPCR was carried out on ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Data were processed by 2-∆∆Ct method and standardized to U6 or GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates.
Western blot
The cultured cardiomyocytes were lysed in RIPA lysis buffer, then separated on 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to PVDF membranes. After being blocked in 5% skim milk, primary antibodies against GAPDH (Cat. #: ab8245; 1/5000, Abcam, Cambridge, UK; control) and ANF (atrial natriuretic factor; Cat. #: ab225844, 1/1000, Abcam), BNP (brain natriuretic peptide; Cat. #: ab92500, 1/10,000 Abcam), β-MHC (β-myosin heavy chain; Cat. #: ab170867, 1/3000, Abcam), p62 (Cat. #: ab109012, 1/30,000, Abcam), LAMP1 (lysosomal associated membrane protein 1; Cat. #: ab108597, 1/5000, Abcam), SIRT1 (Cat. #: ab32441, 1/20,000, Abcam) and USP22 (ubiquitin-specific peptidase 22; Cat. #: ab195289, 1/2000, Abcam) were used. Anti-LC3 (light chain 3) antibody (Cat. #: 14600-1-AP, 1/1500, Proteintech, Rosemont, IL, USA) was procured from Sigma-Aldrich (St. Louis., MO, USA). To be noted, there are two forms of LC3 in various cells, called LC3-I and -II; LC3-I is cytosolic, whereas LC3-II is membrane-bound [28]. After overnight incubation, the HRP-tagged secondary antibodies (Cat. #: ab6728; 1/5000, Abcam) were added for 2 h at room temperature. All protein bands were examined by the ECL detection system (Pierce, Rockford, IL, USA). Western blot results of proteins of interest in the cardiomyocytes were quantified with the help of ImageJ software and statistical analysis was conducted via SPSS version 13.0. Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates.
Subcellular fractionation
In total, 1 × 106 hiPSC-CMs and H9c2 cardiomyocytes were washed in pre-cooled PBS twice for 5 min of centrifugation at 500×g at 4 °C. Cytoplasmic and nuclear fractions were individually extracted using PARIS™ Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s direction. Expression levels of circ-SIRT1 (or circ-Sirt1) in both fractions were assayed by RT-qPCR, with GAPDH and U6 as controls for cell cytoplasm and cell nucleus, respectively. Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates.
Fluorescence in situ hybridization (FISH)
H9c2 and hiPSC-CMs cardiomyocytes were fixed and processed with pepsin, then dehydrated in ethanol. Cells were incubated in hybridization buffer with 40 nM of circ-SIRT1 (or circ-Sirt1)-FISH probe (Ribobio, Guangzhou, China). Following nuclear detection with DAPI solution, cells were detected visually by fluorescence microscope (Olympus Corp., Tokyo, Japan). Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates.
GFP-mRFP-LC3 adenoviral transfection
H9c2 and hiPSC-CMs cardiomyocytes were planted on the glass-bottomed cell culture dishes and then infected with the adenoviral vectors GFP-mRFP-LC3 (Ad-GFP-mRFP-LC3; HanBio Technology, Shanghai, China) expressing the GFP and mRFP fluorescent proteins for marking and tracking LC3 to monitor autophagic influx. Then, the culture medium was changed with the fresh medium, and cells were observed by confocal laser scanning microscope (Zeiss, Dublin, CA, USA) after 24 h to analyze autophagy flux and the number of green, red (autolysosomes), and yellow dots (autophagosomes), which were quantified with the application of Image Plus Pro Software. More than ten cells in three independent bio-repeats were analyzed randomly in the experiment and each bio-repeat contains three technical replicates.
RNA pull-down assays
For RNA-RNA pull-down assays, the cardiomyocytes were subjected to ice-cold PBS and lysis buffer and then incubated with circ-SIRT1 biotin probes (TCTGAAGAGCTCTGTGACCC-biotin) or NC-biotin probes (AGACTTCTCGACTGTGACCC-biotin) at room temperature for 2 h. After the addition of streptavidin magnetic beads (Invitrogen, Carlsbad, CA, USA), the biotin-coupled circ-SIRT1 complex was incubated for another 4 h at 4 °C, followed by washing with RIP washing buffer. Subsequently, the binding miRNAs were extracted from the pulldowns using TRIzol and detected using qRT-PCR. Enrichment values of miRNA (miR-3681-3p, miR-4766-5p, miR-889-3p, or miR-5195-3p) were compared to NC-biotin probe control.
To verify the interaction between miR-3681-3p/miR-5195-3p and circ-SIRT1 and SIRT1, biotinylated miR-3681-3p or miR-5195-3p (Bio-miR-3681-3p: acacagugcuucauccacuacu-biotin; Bio-miR-5195-3p: auccaguucucugagggggcu-biotin) was incubated with streptavidin magnetic beads. Then, circ-SIRT1 and SIRT1 in the pulldowns were isolated using TRIzol and subjected to qRT-PCR analysis. Enrichment values of circ-SIRT1/SIRT1 were relative to Bio-NC control, and the data were mean ± standard variation (SD) of three replicates. For RNA-protein pull-down assays, Pierce Magnetic RNA-Protein Pull-Down Kit was acquired from Thermo Fisher (Waltham, MA) for RNA-protein pull-down assay in cardiomyocytes. The protein extracts were prepared and set as three groups, including Input, circ-SIRT1 biotin probe, and circ-SIRT1 no-biotin probe. The protein extracts were mixed with the biotin-tagged circ-SIRT1 probes. The magnetic beads were added for 1 h. Following RNA-protein pull-down assay, the circ-SIRT1 interacting proteins were subjected to SDS-PAGE and silver staining, followed by mass spectrometry analysis (CapitalBio Technology, Beijing, China) of the specific band. Besides, the enriched USP22 was detected by western blot. Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates.
RNA immunoprecipitation (RIP)
Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit was available for RIP assay in 1 × 107 cardiomyocytes as required by the supplier (Millipore, Bedford, MA, USA). Cell lysates were incubated with RIP buffer adding the magnetic beads and specific antibodies to human Ago2 (Cat. #: 2897; 1/50, Cell Signaling Technology) and USP22 (Cat. #: ab195289; 1/40, Abcam). Normal mouse IgG antibody (Cat. #: 3420; 1/20, Cell Signaling Technology) was used in the control group. Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates.
Co-immunoprecipitation (CoIP)
The cardiomyocytes were harvested by centrifugation at 400×g for 3 min. The processed cardiomyocytes were re-suspended in IP lysis buffer and subsequently left on ice for 15 min. After that, the cardiomyocytes were sonicated for 2 × 10 s and placed directly on ice, followed by centrifugation at 10,000×g for 10 min at 4 °C. Afterward, the supernatant was transferred to a fresh cold Eppendorf tube. Bradford assay was applied to determine the protein concentration. The lysate was incubated with the specific primary antibodies against myc (Cat. #: 2276; 1/1000, Cell Signaling Technology), Flag (Cat. #: 14793; 1/50, Cell Signaling Technology), SIRT1 (Cat. #: 8469; 1/100, Cell Signaling Technology), USP22 (Cat. #: ab195289, 1/40, Abcam), c-Myc (Cat. #: ab32072; 5 µg/ml, Abcam), Ub (ubiquitin; Cat. #: 62802; 1/100, Cell Signaling Technology), or IgG antibody (Cat. #: 3420; 1/20, Cell Signaling Technology) plus A/G sepharose beads or A/G sepharose beads only on a rotator overnight at 4 °C. Then, cell samples were washed in IP lysis buffer and boiled in 4× SDS-loading buffer for western blot analysis. Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates.
Luciferase reporter assay
The miR-3681-3p target sequences (wild-type or mutated) within the SIRT1 fragment were inserted in pmirGLO luciferase vector (Promega, Madison, WI), named as SIRT1-WT and SIRT1-Mut vectors. After co-transfection with NC mimics, miR-3681-3p mimics or miR-3681-3p mimics + circ-SIRT1-oe, cardiomyocytes were assayed with Luciferase Reporter Assay System (Promega). In addition, cardiomyocytes were co-transfected with circ-SIRT1-oe or NC-pcDNA3.1(+), and pGL3 vector containing SIRT1 promoter or pmirGLO vector covering SIRT1 3’UTR for luciferase assay. Three independent bio-repeats were conducted in the experiment and each bio-repeat contains three technical replicates.
Statistical analyses
Data were analyzed by SPSS version 13.0 (SPSS, Chicago, IL) and displayed as the mean ± SD. Experimental results were analyzed by one-way or two-way analysis of variance (ANOVA) or Student’s t test, with two-tailed P value below 0.05 as a significant level.
