Animal model and experimental groups design
The experimental animal model performed was according to the NIH Guide for the Care and Use of Laboratory Animals. The protocols were approved by Institutional Animal Care and Use Committee of Hualien Tzu chi hospital, Taiwan (IACUC approval No. 109-02). Six weeks old Sprague Dawley (SD) rats of 230-255 g were acclimatized for two weeks in the core facility, and thereafter used for experiments. All the rats were housed at a constant temperature (22°C) on a 12-h light-dark cycle with access to diet and water (Lab Diet 5001; PMI Nutrition International Inc., Brentwood, MO, USA). All the rats were arranged into six different groups: control SD rats (n=6), STZ-induced diabetes rats administered with streptozotocin injection (n=5) (55 mg/kg body weight and STZ was dissolved in citrate buffer with pH 4.5) via intraperitoneal cavity, STZ-induced diabetes rats transplanted with WJMSCs (n=6) (1 x 107), STZ-induced diabetic rats infused with GFP-CHIP overexpressed WJMSCs (n=6) (1 x 107), STZ-induced diabetes rats transplanted with WJMSCs containing shCHIP (n=5) (1 x 107), and STZ-induced diabetic rats injected with shPTEN WJMSCs (n=5) (1 x 107). The WJMSCs alone, and WJMSCs expressing lentiviral GFP-CHIP, shCHIP and shPTEN were injected twice via lateral tail vein.
Establishment of stable cell line
The lentiviral plasmids, including GFP-CHIP, shCHIP, and shPTEN were co-transfected with pMD.G and pCMVΔR8.91 plasmids in HEK 293T cell line. The medium was harvested from the 293T cells after 24 and 48 h post transfection. After the lentivirus packaging in the 293T, WJMSCs were infected using polybrene (10 μg/ml). After 48 h, the normal medium was replaced with the medium containing puromycin (5 μg/ml). Thereafter, the cells were harvested and used for experiments.
Oral glucose tolerance test (OGTT)
After six weeks treatment, OGTT was performed to assess insulin resistance. Briefly, rats were fasted for 14 h followed by glucose administration (2 g/kg body weight) using oral gavage method. Blood glucose was measured at the indicated time points (0, 30, 60, 90, 120) by tail vein pricking method using Accu-Chek Guide blood glucose meter (Roche diabetes care, Mannheim, Germany).
Echocardiography imaging was performed to evaluate the cardiac function following the instructions issued by American Society of Echocardiography using a 5-8 MHz sector and 12 MHz linear transducer ((Vivid 3, General Electric Medical Systems Ultrasound, Tirat Carmel, Israel). Briefly, rats were anesthetized, and M-mode as well as two dimensional images were obtained in the parasternal long and short axes. The cardiac parameters including left ventricular diameter (LVD), interventricular septal thickness (IVS), and left ventricular posterior wall thickness (LVPW) were obtained during systole (s) and diastole (d). EF and FS were based on the values as shown in the echocardiography images.
Hematoxylin and eosin (HE), Masson’s trichrome, (MT) and Periodic acid–Schiff staining (PAS)
The tissues slides were deparaffinized using xylene followed by rehydration via gradient alcohol series. All the tissue sections were incubated with HE, MT, and PAS staining dye and subsequently washed with the water. Then, the animal tissues were dehydrated using gradient alcohol series, soaked in xylene, and mounted. Finally, images were obtained using microscopy (OLYMPUS® BX53, Tokyo, Japan).
Immunohistochemical staining (IHC)
As mentioned above, the cardiac tissue sections were deparaffinized with xylene and rehydrated using graded series of alcohol followed by permeabilization, blocking, and washed with PBS. Then, the tissue slides were probed with the respective primary antibody for 1 h, washed with PBS, and incubated with the horseradish peroxidase-conjugated avidin biotin complex using Vectastain Elite ABC Kit and NovaRED chromogen (Vector Laboratories, Burlingame, CA,USA) followed by hematoxylin stain. Expression of cardiac PTEN and FOXO3a was measured using microscopy (OLYMPUS® BX53, Tokyo, Japan).
Reagents and antibodies
All chemicals and reagents were procured from Sigma Aldrich (Sigma Aldrich, St. Louis, USA) unless and otherwise mentioned. Plasmid with the backbone of pRK5 expressing HA tag encoding CHIP was gifted from Dr. Jeng‐Fan Lo (National Yang‐Ming Medical University, Taipei, Taiwan). Lentiviral GFP-CHIP was purchased from the Sino biological (RG83573-ACGLN) (Beijing, China), while the lentiviral expressing small hairpin RNAs (shRNAs), including shcontrol (pLAS.Void), shCHIP (TRCN0000007528 NM_005861), shPTEN (TRCN0000002746 NM_000314), and lentiviral packaging plasmids (pCMVΔR8.91 and pMD.G) were obtained from the national RNAi core facility (academia sinica, Taipei, Taiwan). PTEN inhibitor, (VO-OHpic trihydrate, sc-216061) was purchased from Santa Cruz, Biotechnology (CA, USA), while the HSP90 inhibitor geldanamycin (GA) was bought from Biosciences.
The primary antibodies used in this study are; anti-CHIP (sc-66830), anti-Bcl-2 (C-2) (sc-7382), anti-Bax (P-19) (sc-526), anti-Bad (C-7) (sc-8044), anti-cytochrome C (7H8) (sc-13560), anti-Bcl-xL (H-5) (sc-8392), anti-NOX-2/gp91 phox (sc-5827), anti-p47phox (sc-14015), anti-p22phox (FL-195) (sc-20781), anti-SOD-2 (MnS-1) (sc-65437), anti-catalase (H-9) (sc-271803) anti-Akt1 (B-1) (sc-5298), anti-p-Akt1/2/3 (Ser473) (sc-7985), anti-Bim (H-5) (sc-374358), anti-HA (sc-7392), anti-GFP (FL) (sc-8334), anti-ubiquitin (sc-8017), anti-HDAC1 (sc-81598), anti-β-actin (sc-47778), anti-GAPDH (6C5) (sc-32233) (Santa Cruz Biotechnology, CA, USA), anti-PTEN (#9559s), anti-FOXO3a (75D8) (#2497), anti-p-FOXO3a (Ser253) (#9466), anti-HSP70 (#4872), anti-HSP90 (#4877), (Cell Signaling Technology, MA, USA), and anti-Rac1 (ab33186, abcam, MA, USA). The secondary antibodies against goat, mouse, and rabbit conjugated with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (CA, USA).
Cell culture, transient transfection, and gene silencing
WJMSCs purchased from Bioresource Collection and Research Center (BCRC, Taipei, Taiwan) were grown and maintained in 5% CO2 humidiﬁed incubator (Thermo Fisher Scientific, NY) at 37°C in Dulbecco's Modiﬁed Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, CA, USA), 2 mM glutamine, 1.5 g/L sodium bicarbonate, 100 U/ml penicillin, and 100 mg/ml streptomycin. Briefly, WJMSCs with 50–70% confluency were challenged with HG (40 mM) for 24 h, followed by plasmids and/or siRNA transfection for 24 h using JetPrime transfection reagent (Polyplus-transfection, New York, USA) according to the manufacturer instructions. In this study, MG-132 (proteasome inhibitor), cycloheximide (CHX) (protein synthesis inhibitor), VO-OHpic trihydrate (PTEN inhibitor), and LY294002 (PI3K inhibitor) were treated in the presence of HG.
Western blotting and Immunoprecipitation
Western blot analysis was performed as described in our recent studies [35, 36]. In brief, WJMSCs were centrifuged at 13,000 × g for 20 min after lysed with lysis buffer (50 mM Tris-base, 1 M EDTA, 0.5 M NaCl, 1 mM beta-mercaptoethanol, 1% NP-40, protease inhibitor tablet (Roche, Manheim, Germany) and 10% glycerol). Thereafter, total cell extract was quantified using bradford assay (Bio-Rad, CA, USA), separated by 10–12% SDS‐PAGE, and then transferred to a PVDF membrane (Millipore, MA, USA). Then, membrane was blocked for 1 h in 5% blocking buffer (skim‐milk) followed by overnight incubation in primary antibodies at 4°C. In the next step, membrane was incubated with secondary antibodies (1:3,000 dilution) conjugated with HRP for 1 h at room temperature (RT). Finally, the analysis was obtained using enhanced chemiluminescence (ECL) kit (Millipore, MA, USA), and visualized with LAS 3000 imaging system (Fujifilm, Tokyo, Japan). All the images were quantified and analyzed using ImageJ (NIH, Bethesda, MD, USA) and GraphPad prism5 software respectively.
Whole cell lysates from the WJMSCs were immunoprecipitated using the Protein G magnetic beads (Millipore) following the manufacturer’s guidelines. A total of 500 μg protein lysates were incubated with the 2 μg of respective primary antibody overnight on a rotator at 4°C. Immunoprecipitated proteins were eluted at 95°C and thereafter separated using SDS-PAGE followed by transfer to a PVDF membrane, and probed with specific primary antibody.
Cell viability assay
A colorimetric assay was performed to estimate the cell viability on the principle of conversion tetrazolium (MTT) dye (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide) into a formazan product with blue color formation. After harvest, cells were washed twice with PBS and cultured in DMEM (1 ml) with 10% FBS. MTT (0.5 mg/ml) was added to cells for 4 h and kept at 37°C. Cell viability was measured at OD 570 nm spectrophotometrically after incubation and shaking for 10 min in DMSO.
The cytoplasmic and nuclear extracts were obtained after transfection with siCHIP in the presence of HG stress using Nuclear and Cytosol fractionation kit (BioVision, CA, USA) following the manufacturer's instructions. Briefly, 30-40 μg of separated proteins were analyzed via immunoblotting according to the standard described method.
Detection of Mitochondrial ROS
Mitochondrial superoxide generation was measured in WJMSCs and H9c2 cells using Mitosox (Invitrogen Molecular Probes). After WJMSCs were transfected and challenged with HG for 24 h, cells were incubated with Mitosox for 30 minutes at 37°C, followed by DAPI for 5 min to examine the cell nuclei. Mitochondrial ROS generation was measured using fluorescence microscopy (Olympus, Tokyo Japan), with the excitation and emission wavelength in the range of 510/580 nm.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
After CHIP plasmid transfection and challenged with HG, cells were fixed with 4% Paraformaldehyde for 1 h at room temperature. After washing with PBS, cells were permeabilized with Triton X‐100 (0.1%) in sodium citrate (0.1%), and incubated with TUNEL reagent to measure apoptosis using apoptotic detection kit (Roche Diagnostic, Penzberg, Germany). In cardiac tissue, slides were deparaffinized, rehydrated followed by incubation with 3% H2O2. Thereafter, sections were washed, and incubated with TUNEL reagent for 1 h at 37 °C. Next, cells were incubated with DAPI for 5 min, followed by washing with PBS. Finally, apoptosis was examined by detecting the TUNEL-positive cells using fluorescence microscopy (Olympus, Tokyo, Japan) having excitation and emission wavelength of 450-500 nm and 515-565 nm respectively. The number of TUNEL-positive cells counted manually and statistically analyzed using GraphPad Prism5 software.
The CHIP and PTEN sequences from Homo sapiens were submitted to SBASE server (http://pongor.itk.ppke.hu/protein/sbase.html#/sbase) for domain prediction and structures were collected from PDB database (https://www.rcsb.org/). Active site of PTEN and CHIP were identified using CASTp server (http://sts.bioe.uic.edu/castp). CHIP is docked into the active site of PTEN, and the interaction of CHIP with the active site residues are thoroughly studied using calculations of molecular mechanics using GOLD 3.0.1 software. The default algorithm speed was selected, and the inhibitor binding site in PTEN was defined within a 10Å radius with the centroid as HH atom of SER94 in Homo sapiens respectively. After docking, the individual binding poses of CHIP was observed and interaction with the PTEN were studied. The best and most energetically favorable conformation of CHIP was selected .
Bim promoter region was collected from NCBI database, (https://www.ncbi.nlm.nih.gov/) and drawn using Avogadro software which was a molecule generator algorithm. Later the FOXO3a structure collected from the PDB database was docked with Bim promoter sequence using GOLD 3.0.1. The binding studies of Bim promoter with FOXO3a protein predicted to find the interaction sites of FOXO3a with the Bim promoter region .
Flow cytometry for apoptosis detection
Cells transfected with HA-vector, HA-CHIP, shcontrol, and/or shCHIP plasmid in the presence of HG were harvested, and washed twice with PBS. Then, cells were resuspended in 1x binding buffer, and incubated for 15-30 min with isothiocyanate (FITC) annexin V fluorescein and propidium iodide (PI) dye using FITC Annexin V detection kit (BD, Biosciences, CA, USA) following the manufacturer instructions and analyzed by Fluorescence Activated Cell Sorting (FACS) (BD, Biosciences). The statistical analyses were based on the 10,000 cells per event.
WJMSCs and H9c2 cardiomyocytes were co-cultured in 6-well Transwell inserts (Corning, USA) with 0.4 μm pore size, and maintained in a 5% CO2 humidified incubator. WJMSCs alone, WJMSCs expressing HA-CHIP, HA-vector, sicontrol, siCHIP, and/or CHIP mutants (K30A and H260Q) were seeded in the inner transwell chamber, while H9c2 cells in the lower chamber were challenged with HG.
Results are shown as mean ± SD. Statistical analysis was performed using GraphPad Prism5 statistical software. Multiple comparisons were accessed through one‐way ANOVA and p-Value of <0.05 was considered statistically signiﬁcant.