CHIP Reverses Hyperglycemia-associated PTEN Stability in Wharton's Jelly Derived Mesenchymal Stem Cells and Promotes Its Therapeutic Potential Against Diabetes-induced Cardiac Injury

Recent studies indicate that umbilical cord stem cells are cytoprotective against several disorders. One critical limitation in using stem cells is reduction in their viability under stressful conditions, such as diabetes. However, the molecular intricacies responsible for diabetic conditions are not fully elucidated. Effects of HG on Wharton's jelly derived mesenchymal stem cells (WJMSCs) viability was evaluated by MTT assay and ow cytometry. The mechanism responsible for HG-induced PTEN degradation was assessed using loss and gain of function, immunouorescence, co-immunoprecipitation, and western blot analysis. Co-culturing of CHIP-overexpressed WJMSCs with embryo derived cardiomyoblasts was performed to analyze their ameliorative effects. The therapeutic effects of CHIP expressing WJMSCs were further validated in Sprague Dawley male (eight weeks old) STZ-induced diabetic animals by echocardiography, immunohistochemistry, hematoxylin eosin, and masson’s trichrome and TUNEL staining. Multiple comparisons were accessed through one ‐ way ANOVA and p-Value of <0.05 was considered statistically signi ﬁ cant. CHX for using blot analysis. (I) WJMSCs transfected with HA-vector, HA-CHIP K30A) challenged with HG for 24 h, and viability using MTT assay. WJMSCs were transfected with pRK5-HA-vector pRK5-HA-CHIP pRK5-HA-H260Q by HG 24 lysates

Diabetic patients exhibited an increased risk of heart failure [1][2][3]. Lack of insulin, pregnancy or insulin resistance may lead to the hyperglycemic condition [4]. Hyperglycemia can induce apoptosis in many tissues and cells [5,6] that triggered the generation of reactive oxygen species (ROS), cardiac apoptosis, leading to the complication of diabetic cardiomyopathy [7,8]. Diabetic cardiomyopathy is a condition associated with abnormal ventricular function in diabetic patients in the absence of any other risk factors, such as hypertension and coronary atherosclerosis that occurs as a result of abnormal lipid and glucose metabolism resulting in the elevation of oxidative stress and other signaling cascades [9][10][11].
Number of studies has shown that hyperglycemic conditions, such as diabetes induced apoptosis, senescence, and reduce the proliferation ability of mesenchymal stem cells [12][13][14]. According to the previous study, diabetic condition limit survival of the transplanted stem cells by initiating apoptosis leading to cell death [15]. Insulin, hypoglycemic agents, and dietary control are the currently available therapeutic strategies using worldwide for diabetes and its associated complications have limitations [16]. Therefore, there is a need for cell-based therapy to overcome this problem.
Phosphatase and tensin homolog (PTEN) is a tumor suppressor protein, encoded by the PTEN gene. It is widely accepted that the level of PTEN should be tightly regulated as it involved in numerous cellular processes. NEDD4-1, XIAP, and WWP2 are E3 ubiquitin ligase that maintains the level of PTEN via the ubiquitin-proteasome system. Among them, NEDD4-1 is the rst identi ed E3 ligase that polyubiquitinates PTEN, which results in PTEN degradation [17,18]. PTEN negatively regulates the PI3K/Akt signaling pathway by converting PIP3 back to PIP2 [19,20]. Previous study demonstrated that inhibition of PTEN reverses the hyperglycemic effects in mice [21]. In another study, it was revealed that change in PTEN expression level regulates the muscle protein degradation in diabetic mice [22]. Besides, there is not enough evidence about the increase or change in the localization of PTEN in NEDD4-1 knockout cells, suggesting the involvement of other E3 ligases that may target PTEN for ubiquitylation [19,23].
The Carboxyl terminus of Hsc70 interacting protein (CHIP) cDNA encodes a 34.5 kDa well-conserved protein that has around 98% sequence similarity with the mouse and ∼60% similarity with the fruit y [24]. CHIP contains a conserved U-box domain at their C terminus having E3 ligase activity and an N terminal tetratricopeptide repeat (TPR) domain responsible for interaction with Hsc/p70 and HSP90 clients [25,26]. An earlier study demonstrated the protective effect of CHIP against myocardial injury induced apoptosis and oxidative stress in the CHIP-su cient animal model [27]. A recent study from our lab highlighted the protective effect of CHIP against doxorubicin induced cardiomyocyte death [28]. In another study, we have envisaged that isoproterenol induced cytotoxicity attenuated by Tid-1s through CHIP mediated Gαs degradation [29]. However, to the best of our knowledge, it is not fully elucidated that whether CHIP could exert a protective effect against hyperglycemia-induced apoptosis and oxidative stress under diabetic condition.
Wharton's jelly derived mesenchymal stem cells (WJMSCs) are the broblast-like, highly homogenous population of cells that differ it from other stem cell sources [30,31]. The unique characteristics of WJMSCs are therapeutic potential, immune privilege, ease of isolation, and multi-differentiation potential [32]. These properties make the WJMSCs as an ideal source for the treatment of many organs [33]. The stem cells has the differentiation ability which can replace the dead cells, and release certain factors that trigger nearby cells in the microenvironment to accelerate the repair process [34]. However, much less attention has been paid to elucidate whether WJMSCs have therapeutic potential against diabetes induced cardiac damages.
Research has shown that HG-induced apoptosis and oxidative stress exerts a negative effect on stem cell function. However, the underlying mechanism that attenuates HG-induced apoptosis and oxidative stress in diabetes remains elusive. In this study, we hypothesized that CHIP overexpressing WJMSCs may prevent HG-induced apoptosis and oxidative stress by promoting ubiquitin-mediated proteasomal degradation of PTEN, and might exert therapeutic effects against diabetes-associated cardiac damage.

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 10 7 ), STZinduced diabetic rats infused with GFP-CHIP overexpressed WJMSCs (n=6) (1 x 10 7 ), STZ-induced diabetes rats transplanted with WJMSCs containing shCHIP (n=5) (1 x 10 7 ), and STZ-induced diabetic rats injected with shPTEN WJMSCs (n=5) (1 x 10 7 ). 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. Brie y, 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
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). Brie y, 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 depara nized 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 depara nized 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).

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 quanti ed 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 (Fuji lm, Tokyo, Japan). All the images were quanti ed 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 speci c 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.

Subcellular fractionation
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. Brie y, 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 uorescence 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 xed 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 depara nized, rehydrated followed by incubation with 3% H 2 O 2 . 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 uorescence 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.

Insilico analysis
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 identi ed 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 de ned 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 [37].
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 nd the interaction sites of FOXO3a with the Bim promoter region [38].

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 uorescein 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.

Statistical analysis
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.

HG induces apoptosis and oxidative stress via activation of PTEN and the downstream signaling cascade in WJMSCs
Previous studies have demonstrated that HG induces apoptosis and oxidative stress in various cell lines. Considering these, rstly, we assessed the effect of HG on cell viability. We observed that the cell viability was considerably reduced in a time-and dose-dependent manner in WJMSCs after being challenged with HG ( Fig. 1A). Thereafter, we assessed whether HG can induce apoptosis in WJMSCs using ow cytometry and western blot. Results indicated that increasing HG concentrations reduced the percentage of viable cells whereas the percentage of apoptotic cells (early, late) was signi cantly elevated in a dose-dependent manner (Fig. 1B). HG is the key regulator of mitochondrial ROS generation. Interestingly, immuno uorescence microscopy imaging showed increased ROS production with increasing HG concentrations (Fig. 1C). These results indicate that HG can reduce cell viability and induce apoptosis and oxidative stress in a dose-dependent manner in WJMSCs. In earlier studies, it was reported that HGinduced oxidative stress and apoptosis are regulated via the FOXO3a pathway [39]. Therefore, we evaluated the impact of HG on PTEN and the downstream signaling cascade in WJMSCs. Following HG induction, western blot data demonstrated that PTEN expression increased with concomitant impairment in p-AKT and elevation of FOXO3a and the downstream regulator (Bim) in a dose-dependent manner (Figs. 1D, E). Further, it was found that PTEN levels were considerably reduced in a time-dependent manner following cycloheximide (CHX) treatment; however, treatment with CHX in the presence of MG-132 and HG conditions stabilized their expression level, indicating the ine cient degradation of PTEN (Fig. 1F). The above data suggest that the PTEN/FOXO3a/Bim signaling pathway may be involved in HGinduced apoptosis in WJMSCs.

CHIP overexpressed WJMSCs attenuate HG-induced PTEN-mediated apoptosis and oxidative stress
Earlier studies demonstrated the protective role of CHIP against various stress conditions. Therefore, the molecular mechanism responsible for CHIP-induced apoptosis and oxidative stress resistance was evaluated in WJMSCs. The expression of chaperones was analyzed after HG administration. Western blot analysis revealed that the expression of the chaperone system, including CHIP, HSP90, and HSP70 was reduced as compared to controls (Fig. 1A). Further, cell viability was retained dose dependently in CHIP overexpressing cells after HG induction (Figs. 2B). Moreover, it was found that enhanced CHIP expression attenuated PTEN expression level, rescued AKT levels, which were impaired under HG conditions, promoted the phosphorylation of FOXO3a, and inhibited the binding of FOXO3a with Bim in a dosedependent manner (Fig. 2C). Next, we evaluated whether CHIP can attenuate HG-induced apoptosis and mitochondrial ROS generation under HG conditions. Flow cytometry and mitosox staining ascertained that CHIP overexpression suppressed apoptosis and mitochondrial ROS dose dependently, as compared to HG (Figs. 2D, E). Furthermore, PTEN levels were not reduced to a considerable extent in the presence of MG-132 (proteasome inhibitor), which suggests that CHIP may target PTEN for proteasomal degradation (Fig. 2F). CHIP knockdown, using the siRNA approach, showed moderate activity to inhibit PTEN in the presence of the proteasome inhibitor (Fig. 2G). Thereafter, we evaluated the in uence of CHIP overexpression and knockdown on endogenous PTEN levels under HG conditions, followed by cycloheximide treatment. The half-life of PTEN was increased and decreased after transfection with siCHIP or CHIP, respectively (Fig. 2H). Additionally, cell viability was retained in wild type CHIP compared to the HG group, whereas no considerable effects were seen in CHIP mutant groups (Fig. 2I). We further assessed whether CHIP has the potential to attenuate the expression of PTEN and inhibit its downstream signaling cascade in the presence and absence of MG-132 under HG stress. For this, we employed various CHIP mutants, namely K30A (TPR domain mutant) and H260Q (U-box domain mutant), together with wild type CHIP. Notably, it was found that wild type CHIP suppressed PTEN and FOXO3a expression, leading to the induction of p-AKT and p-FOXO3a protein expression levels. However, the vector and CHIP mutants (K30A and H260Q) did not exhibit any effects (Figs. 2J, S2A). Collectively, this data demonstrates that CHIP suppressed HG-induced PTEN expression, reduced its half-life, and prevented apoptosis and oxidative stress; nevertheless, neither of the CHIP mutants (H260Q and K30A) were able to attenuate PTEN in WJMSCs. The CHIP and PTEN structures were collected from PDB database using IDs 4KBQ and 1D5R, respectively ( Figures S2B, S2C). In the SBASE search results, CHIP displayed three TPR domains (26-59, 60-93, and 94-127) and one U-box domain (226-300). In PTEN, three different domains, namely dual speci city protein phosphatase , cyclic nucleotide binding (219-261), and ferredoxin like domain (377-395), were predicted by the SBASE server. The possible binding sites of CHIP and PTEN were investigated using the CASTp server, and residues are shown in Figure S2D and S2E ( Figures S2D, S2E). Multiple prediction methods were used to identify binding sites based on structure. The docking method with GOLD 3.0.1 predicted ten con rmations and binding scores. Our analysis revealed that the N-terminal of the CHIP domain has a high tendency for the N-terminal of the PTEN domain, and are potentially involved in binding. The total clusters of docking conformations with the docked PTEN showed positive binding energies. Among all docking conformations, the best predicted binding free energy is -125.34 KJ/mol to PTEN ( Figure S2F).

CHIP regulates PTEN and its downstream signaling cascade under HG conditions in WJMSCs
CHIP can downregulate PTEN, thereby inhibiting HG-induced apoptosis and oxidative stress. Therefore, we evaluated the impact of CHIP knockdown on cell viability and PTEN and its downstream signaling cascade under HG conditions. The cell viability was reduced dose dependently upon CHIP inhibition, and showed a signi cant increase in PTEN, FOXO3a, and Bim protein expression levels in a dose-dependent manner, as compared to control and HG condition alone. However, the expression of p-AKT and p-FOXO3a was downregulated in the presence of siCHIP (Figs. 3A, B). Next, we determined the effect of CHIP inhibition on apoptosis and oxidative stress. Flow cytometry and mitosox staining revealed that the percentage of apoptotic cells (early, late) and mitochondrial ROS generation was elevated in a dosedependent manner upon CHIP knockdown, following HG induction (Figs. 3C, D). We further evaluated the effect of PTEN inhibition on the downstream signaling pathway. The protein expression of p-AKT appeared increased, while the FOXO3a and Bim interaction was inhibited in a dose-dependent manner after HG exposure (Fig. 3E). CHIP knockdown elevated the expression of PTEN and the downstream signaling mediators, which indicates that CHIP regulates the PTEN/FOXO3a/Bim signaling pathway (Fig. 3F). We further validated the effects of CHIP on apoptosis and oxidative stress under HG conditions. The data indicates that CHIP overexpression suppressed HG-induced apoptosis and ROS generation, and the effects were reversed upon CHIP knockdown (Figs. 3G, H). We then determined whether CHIP in uences AKT-induced inhibition of FOXO3a under HG conditions. Western blot analysis indicated that CHIP promotes phosphorylation of AKT, which in turn increased p-FOXO3a and decreased Bim expression; however, the effects were reversed upon AKT knockdown (Fig. 3I). In addition, in the presence of siCHIP and/or in the presence of siAkt, similar alterations in the corresponding protein markers were observed, which suggests that CHIP can increase cell survival by regulating the AKT signaling cascade under HG conditions (Fig. 3J). Altogether, these data indicate that CHIP regulates PTEN and the downstream signaling cascade under HG stress in WJMSCs.
CHIP targets PTEN for ubiquitin-mediated proteasomal degradation cooperated by HSP70 under HG conditions Previous data indicate that CHIP overexpression attenuates HG-induced PTEN, and this effect was reversed upon CHIP knockdown. Considering these, we evaluated whether CHIP promotes the ubiquitinmediated proteasomal degradation of PTEN. Co-IP data showed that CHIP directly interacts with PTEN and promotes its ubiquitination (Figs. 4A-C). These results indicate that CHIP has the potential to interact with and promote ubiquitin-mediated proteasomal degradation of PTEN. Further, co-IP was performed to analyze the binding ability and E3 ligase activity of CHIP. The co-IP data revealed that both CHIP mutants have the binding ability, but neither have the potential to ubiquitinate PTEN, which indicates that both domains (K30A and H260Q) are responsible for ubiquitin-mediated proteasomal degradation of PTEN, and the possible involvement of chaperones, as the K30A mutant loses its ability to interact with HSP70 and 90 (Figs. 4D, E). It is well known that CHIP regulates various proteins presented by the HSP70.
Therefore, we evaluated the in uence of HSP70 in CHIP-mediated PTEN degradation. We found that HSP70 inhibition led to a dose-dependent increase in PTEN protein expression (Fig. 4F). Further, HSP70 inhibition blocks the loss of PTEN in the presence of CHIP and/or siCHIP after HG exposure (Figs. 4G, H). Besides, the molecular interaction of HSP70 and PTEN was veri ed using In Silico analysis (Fig. 4I). These data demonstrate that HSP70 co-operates with CHIP to promote PTEN degradation under HG conditions. Collectively, these data suggest that CHIP promotes ubiquitin-mediated proteasomal degradation of PTEN might be co-operated by HSP70 under HG conditions in WJMSCs.
CHIP regulates the binding of FOXO3a with the Bim promoter region FOXO3a, a vital transcription factor can bind to various promoters, including Bim. Therefore, we assessed whether FOXO3a knockdown can in uence the expression of bim under HG conditions. From the immunoblot assay, we observed that Bim was downregulated dose dependently upon FOXO3a knockdown under HG conditions (Fig. 5A). Thereafter, we silenced FOXO3a together with AKT, and observed that AKT inhibition hindered elevation of the pro-apoptotic protein Bim (Fig. 5B). We performed western blotting to further ascertain the effect of CHIP on FOXO3a and its downstream promoter. Western blot analysis revealed that CHIP overexpression inhibited FOXO3a expression and the downstream bim promoter; whereas, CHIP knockdown reversed this effect, indicating that CHIP regulates the binding of FOXO3a with the Bim promoter region via activation of AKT (Fig. 5C). Next, we determined whether CHIP regulates PTEN and FOXO3a protein expression in WJMSCs. As shown in Fig. 5D, HG-induced cytoplasmic PTEN and nuclear FOXO3a protein levels were further increased by silencing CHIP in a dosedependent manner (Fig. 5D). FOXO3a structure was collected from the PDB database (PDB ID: 2K86), con rming the domains fork head transcription factor (148-249) and unknown domain function (373-426) ( Figure S5A). The active site of FOXO3a was predicted using the CASTp server, and includes amino acids TRP157, LEU160, LEU165, ARG168, CYS190, VAL190, and PRO192 ( Figure S5B). FOXO3a binds to the Bim promoter with high a nity and induces apoptosis. The Bim promoter region was selected from the NCBI database and drawn using Avogadro software ( Figure S5C). Docking studies were performed to gain insight into the binding conformation of FOXO3a with the Bim promoter region. All docking calculations were carried out using GOLD and the les generated were analyzed for their binding conformations. Among the active residues, ARG94, ASP95, SER101, TYR102, and SER149 play an important role in forming hydrogen bonds with the Bim promoter region (Fig. 5E). Figure 6A indicates a schematic illustration of the in vitro co-culturing system, in which WJMSCs were cultured in the upper chamber, with cardiac cells in the lower chamber (Fig. 6A). We determined whether CHIP overexpressed WJMSCs could rescue cell viability. The data indicates that co-culturing CHIP overexpressing WJMSCs signi cantly retained the cell viability in H9c2 cells as compared to HG and WJMSCs alone (Fig. 6B). Next, we assessed whether co-culturing of WJMSCs with H9c2 could rescue HGinduced cardiac apoptosis and oxidative stress. Western blot analysis showed that HG considerably induces pro-apoptosis markers, such as Bax, Bad, and Cyt-c, whereas the pro-survival markers, p-AKT and Bcl-xL, were reduced in H9c2 cells. However, co-culturing with WJMSCs, especially CHIP overexpressing WJMSCs, altered HG-induced apoptosis markers in H9c2 cardiomyoblasts. siCHIP approach was used to ascertain the role of CHIP in enhancing the potential of WJMSCs, and it was found that the effects were reversed upon CHIP inhibition compared to the control group, which further validated the role of CHIP in enhancing the potential of WJMSCs (Fig. 6C). These results were further con rmed using ow cytometry. Results indicated that apoptotic cells increased considerably under HG conditions; however, treatment with WJMSCs and CHIP overexpressing WJMSCs considerably reduced the percentage of apoptotic cells (Fig. 6D). Similar results were obtained for oxidative stress markers. The pro-oxidant marker, P22 Phox , was considerably enhanced under HG conditions, whereas the anti-oxidant markers, including SOD-2 and catalase, were reduced. However, co-culturing with WJMSCs, especially CHIP overexpressing WJMSCs, ameliorated HG-induced oxidative stress markers in H9c2 cells. Further, upon knockdown of CHIP, the effects were reversed (Fig. 6E). Moreover, mitosox analysis con rmed the ameliorative effect of CHIP overexpressing WJMSCs against HG-induced oxidative stress (Fig. 6F). In addition, the effect of CHIP mutants was evaluated against cardiac complications. We observed that cardiac apoptosis and oxidative stress markers, such as Bax and P22 Phox , were elevated under HG conditions, whereas the cardioprotective markers, such as Bcl-xL and catalase, were downregulated. Co-culturing with CHIP overexpressing WJMSCs considerably reduced Bax and P22 Phox , while Bcl-xL and catalase were enhanced. However, no effects were observed in CHIP mutant overexpressing cells (Fig. 6G). All these data demonstrate that CHIP overexpressed WJMSCs exert protective effects against HG-induced cardiac apoptosis and oxidative stress.

CHIP overexpressed WJMSCs ameliorated hyperglycemia-induced cardiac damage in STZ-induced diabetic rats
Finally, we assessed whether CHIP overexpressed WJMSCs rescued hyperglycemia-induced cardiac injury in vivo. Before STZ induction, all rats exhibited normal body weight and blood glucose levels (Table 1). Firstly, diabetes was induced using STZ in male SD rats, and after four days animals received WJMSCs alone or WJMSCs carrying lentiviral plasmids, including GFP-CHIP, shCHIP, and shPTEN (Fig. 7A). Oral glucose tolerance test was performed to evaluate the anti-hyperglycemic effects in all experimental groups. STZ-induced diabetes, WJMSCs alone, and shCHIP-WJSMCs groups exhibited markedly increased blood glucose levels and area under the curve (AUC) obtained from OGTT, as compared to the control group. However, CHIP overexpressed WJMSCs, and shPTEN expressing WJMSCs attenuated the blood glucose levels and AUC as well (Fig. 7B). Moreover, it was observed that whole heart weight (WHW) and left ventricle weight (LVW) were markedly reduced in STZ-induced diabetes, WJMSCs alone, and WJMSCs carrying shCHIP groups. However, CHIP overexpression and PTEN knockdown in WJMSCs signi cantly rescued the WHW and LVW weight in the experimental rats (Fig. 7C). Besides, LVW/WHW, WHW/TL, and LVW/TL exhibited obvious reduction compared to the control group. Interestingly, transplantation of CHIP overexpressed and PTEN knockdown WJMSCs rescued the above-mentioned parameters ( Table 1). Echocardiography was performed to evaluate the cardiac function in experimental rats. The cardiac parameters related to cardiac function were reduced in STZ-induced diabetes, WJMSCs alone, and shCHIP containing WJMSCs as compared to the control group. The ejection fraction (EF) and fractional shortening (FS) were obviously reduced in STZ-induced diabetes, WJMSCs, and shCHIP expressing WJMSCs groups. Moreover, other parameters, such as interventricular septum at diastole (IVSd), internal dimension at diastole of the left ventricle (LVIDd), end-diastolic volume (EDV), stroke volume (SV), and left ventricular diameter mass (LVD) were also lowered in STZ-induced diabetes, and shCHIP carrying WJMSCs groups. Interestingly, infusion of CHIP overexpressed and PTEN silenced WJMSCs signi cantly improved the above-mentioned parameters back to normal levels (Fig. 7D, Table 2). The apoptosis and oxidative stress markers with abnormal protein expression in the left ventricle of STZ, WJMSCs, and shCHIP-WJMSCs reverted to normal levels in the groups with enhanced CHIP and PTENsilenced WJMSCs in the presence of STZ (Fig. 7E). Furthermore, increased interstitial spaces, brosis, collagen, and glycogen accumulation were observed in STZ, WJMSC, and shCHIP-WJSMC groups, but the cardiac damage induced in these groups were rescued after infusion of CHIP overexpressed and PTEN knockdown WJMSCs (Fig. 7F). TUNEL positive cardiac cells induced by STZ-induced diabetes, WJMSCs, and WJMSCs administered shCHIP were strongly reduced in groups injected with WJMSCs expressing CHIP and shPTEN plasmids (Fig. 7G). Moreover, immunohistochemical imaging ascertained that the expression levels of PTEN and FOXO3a were elevated in groups with STZ, WJMSCs alone, and WJMSCs infused with shCHIP, as compared to controls. In contrast, transplantation of WJMSCs expressing CHIP and shPTEN reduced their expression level (Fig. 7H). These results indicate that CHIP exerts protective effects against hyperglycemia-induced cardiac injury in STZ-induced diabetic rats by reducing PTEN stability in WJMSCs. Collectively, the present study suggests that CHIP targets PTEN for ubiquitin-mediated proteasomal degradation presented by HSP70 under hyperglycemic conditions and further phosphorylates AKT. Moreover, the binding of FOXO3a with Bim was inhibited, resulting in apoptosis resistance (Fig. 7I).

Discussion
Accumulating evidence has highlighted the cytoprotective effects of umbilical cord stem cells against various diseases. Nevertheless, it has also been shown that under stressful conditions, stem cells display reduced potential. An increasing number of studies have shown the adverse effects of hyperglycemic conditions in different stem cells, including mesenchymal stem cells [12][13][14]40]. Taking these into consideration, we rst evaluated the effect of HG on WJMSCs and the underlying mechanism involved in HG-induced cellular injuries in WJMSCs. It was found that HG affects cell viability and affects the components of proteostasis machinery, such as HSP90, HSP70, and CHIP. Moreover, the effect of CHIP overexpression on WJMSCs under HG condition was elucidated further; interestingly, we found that HG activated PTEN and the downstream signaling cascade that ensued in the induction of apoptosis and oxidative stress via CHIP impairment in WJMSCs. Notably, it was found that CHIP targets and promotes proteasomal degradation of PTEN and in turn enhanced p-AKT and p-FOXO3a protein expression levels to inhibit HG-induced apoptosis and oxidative stress. A previous study demonstrated that PTEN is activated during HG conditions and can induce severe cardiac complications, including oxidative stress and apoptosis, leading to diabetic cardiomyopathy [41]. However, the underlying mechanism responsible for regulating PTEN is not fully understood in WJMSCs. It is well known that ubiquitin-mediated proteasomal degradation plays an important role in protein quality control in order to maintain protein homeostasis [42]. Ahmad et al, demonstrated that CHIP promotes the proteasomal degradation of PTEN [43]. In addition, CHIP has the ability to target many proteins for proteasomal degradation [44,45]. Our results are consistent with previous studies, wherein it has been highlighted that CHIP is able to promote the ubiquitination and proteasomal degradation of PTEN, which may be supported by HSP70 and further promotes phosphorylation of AKT and FOXO3a to inhibit HG-induced apoptosis and oxidative stress. Moreover, CHIP overexpression reduced the binding ability of FOXO3a with Bim. In addition, bioinformatics analysis con rmed the interaction of CHIP with PTEN. Docking results indicated that conserved amino-acid residues play an important role in maintaining functional conformation and are directly involved in donor substrate binding. The interaction between CHIP and PTEN proposed in this study is useful for understanding the potential mechanism of domain and inhibitor binding. As is well known, hydrogen bonds play important roles in the structure and function of biological molecules, and we found that PRO102, GLN104, TRP105, and PHE106 in CHIP of Homo sapiens are important for strong hydrogen bonding interaction with THR147, GLY149, and ILE192 of PTEN. To the best of our knowledge, these are conserved in this domain and may be important for structural integrity or maintaining the hydrophobicity of the inhibitor-binding pocket. Collectively, our study increased the understanding of the protective role of CHIP in regulating PTEN and the downstream signaling cascade triggered under HG conditions. As mentioned earlier, several studies demonstrated that FOXO3a, a vital transcription factor involved in many cellular processes, can bind to various promoter regions. Besides, it is well known that bim, a pro-apoptotic protein, can regulate apoptosis under several stress conditions [46,47]. Considering these, it was found that FOXO3a depletion downregulated bim, and similar results were obtained upon CHIP overexpression in WJMSCs, indicating that CHIP regulates binding of FOXO3a with the bim promoter region, which was further con rmed by docking studies. Cumulatively, the present study suggests that CHIP can modulate PTEN and the downstream signaling cascade and confers resistance to apoptosis by promoting PTEN proteasomal degradation.
Diabetes mellitus increases the risk of cardiovascular complications. Evidence has shown that hyperglycemia induces cardiomyopathies. Combining newer therapeutic strategies may provide hope for tackling the devastating complications associated with diabetes, both in the heart, as delineated here, and potentially in other organs as well. As a matter of fact, interest in stem cell-based regenerative medicines has garnered great interest amongst the research fraternities. Interestingly, implementations of genetic engineering methodologies are capable of further enhancing the therapeutic potential of stem cells. Thus, our CHIP overexpressing WJMSCs may be an effective candidate in the frontiers of engineered stem cells with therapeutic potential against HG-induced cardiac injury. Research has highlighted that genetically engineered stem cells have shown e cacy against various diseases [48,49].
Therefore, to ascertain their potential against HG-induced cardiac complications, co-culturing of CHIP overexpressing WJMSCs was performed. Interestingly, it was found that co-culturing of WJMSCs with H9c2 rescued HG-induced apoptosis and oxidative stress. Increasing our understanding may be instrumental in ameliorating stem cell effects and would certainly widen the horizon of stem cell therapeutics. This is indeed in agreement with other studies, wherein the authors have highlighted the potential of various engineered stem cells [50][51][52]. Further, in vivo animal model experimentation was performed to explore the role of CHIP overexpressed WJMSCs against diabetes-induced cardiac injury. Our in vitro ndings are in line with the in vivo model, except that in the animal tissues WJMSCs alone exhibited some effect, which is inconsistent with the cell model ndings. In conclusion, our research demonstrated the underlying intricacies regarding diabetes-induced cellular injuries and provided evidence for the ameliorative effect of CHIP overexpressing WJMSCs against diabetes-induced cardiac complications.

Conclusion
HG increased apoptosis and oxidative stress in cells with impaired proteostasis systems, which trigger the PTEN signaling cascade. CHIP, an E3 ligase, maintains PTEN under HG conditions via association with the chaperone system. Furthermore, CHIP knockdown stabilizes PTEN; while CHIP overexpression induces Akt and promotes the phosphorylation of FOXO3a, resulting in export from the nucleus to the cytoplasm, and inhibits the binding of FOXO3a with the Bim promoter. Moreover, the co-culturing of CHIP overexpressing WJMSCs with H9c2 rescued HG-induced apoptosis and oxidative stress, and its administration to STZ-induced diabetic rats attenuated cardiac damage. Cumulatively, the present study reveals that CHIP overexpressing WJMSCs promote apoptosis resistance through alteration of PTEN and the downstream signaling cascade, and more speci cally, by promoting PTEN proteasomal degradation.

Not applicable
Availability of data and materials The datasets generated or analyzed in this study are available from the corresponding author on reasonable request.