E3 ligase activity of Carboxyl terminus of Hsc70 interacting protein (CHIP) in Wharton's jelly derived mesenchymal stem cells improves their persistence under hyperglycemic stress and promotes the prophylactic effects against diabetic cardiac damages

Abstract 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. In this study, we found that high glucose (HG) conditions induced loss of chaperone homeostasis, stabilized PTEN, triggered the downstream signaling cascade, and induced apoptosis and oxidative stress in Wharton's jelly derived mesenchymal stem cells (WJMSCs). Increased Carboxyl terminus of Hsc70 interacting protein (CHIP) expression promoted phosphatase and tensin homolog (PTEN) degradation via the ubiquitin‐proteasome system and shortened its half‐life during HG stress. Docking studies confirmed the interaction of CHIP with PTEN and FOXO3a with the Bim promoter region. Further, it was found that the chaperone system is involved in CHIP‐mediated PTEN proteasomal degradation. CHIP depletion stabilizes PTEN whereas PTEN inhibition showed an inverse effect. CHIP overactivation suppressed the binding of FOXO3a with bim. Coculturing CHIP overexpressed WJMSCs suppressed HG‐induced apoptosis and oxidative stress in embryo derived cardiac cell lines. CHIP overexpressing and PTEN silenced WJMSCs ameliorated diabetic effects in streptozotocin (STZ) induced diabetic rats and further improved their body weight and heart weight, and rescued from hyperglycemia‐induced cardiac injury. Considering these, the current study suggests that CHIP confers resistance to apoptosis and acts as a potentiation factor in WJMSCs to provide protection from degenerative effects of diabetes.


| INTRODUCTION
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.
Wharton's jelly derived mesenchymal stem cells (WJMSCs) are the fibroblast-like, highly homogenous population of cells that differ it from other stem cell sources. 17,18 The unique characteristics of WJMSCs are therapeutic potential, immune privilege, ease of isolation, and multidifferentiation potential. 19 These properties make the WJMSCs as an ideal source for the treatment of many organs. 20 The stem cells have 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. 21 The effect of WJMSCs on diabetes has been widely investigated however, much less attention has been paid to elucidate whether WJMSCs have therapeutic potential against diabetes induced cardiac damages. Moreover, the reduction in the survival of the transplanted stem cells under hyperglycemic condition still remains as a concern for their clinical use.
Glucose intolerance is one of the phenomena often associated with diabetes particularly in the Type II diabetes. Conditions like glucose intolerance and obesity are often associated with upregulated levels of phosphatase and tensin homolog (PTEN). 22 PTEN regulation is often associated with maintenance of survival mechanism and tissue homeostasis in various tissues, such as heart. Since maintenance of the survival and proliferation potential of stem cells are crucial criteria for a success transplantation therapy, PTEN knockdown is correlated with better stem cell function and repair. 23 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 first identified E3 ligase that polyubiquitinates PTEN, which results in PTEN degradation. 24,25 PTEN acts as a tumor suppressor protein that negatively regulates the PI3K/Akt signaling pathway by converting PIP3 back to PIP2. 26,27 Previous study demonstrated that inhibition of PTEN reverses the hyperglycemic effects in mice. 28 In another study, it was revealed that change in PTEN expression level regulates the muscle protein degradation in diabetic mice. 29 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. 26,30 Therefore in this study, we identify the PTEN regulating chaperone that help in controlling the PTEN expression levels in order to improve the functions of WJMSCs and maintain tissue homeostasis in diabetic conditions. 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 fly. 31 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. 32,33 An earlier study demonstrated the protective effect of CHIP against myocardial injury induced apoptosis and oxidative stress in the CHIP-sufficient animal model. 34 A recent study from our lab highlighted the protective effect of CHIP against doxorubicin induced cardiomyocyte death. 35 In another study, we have envisaged that isoproterenol induced cytotoxicity attenuated by Tid-1s through CHIP mediated Gαs degradation. 36 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.
Research has shown that high glucose (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.

| 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, first, 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 (Figure 1(a)). Thereafter, we assessed whether HG can induce apoptosis in WJMSCs using flow cytometry and Western blot. Results indicated that increasing HG concentrations reduced the percentage of viable cells whereas the percentage F I G U R E 1 Effect of HG on PTEN-mediated apoptosis and oxidative stress in WJMSCs. (a) WJMSCs seeded under varying concentration of HG for the indicated time points (24,48, and 72 h) were harvested, and the cell viability was performed. (b, c) WJMSCs challenged with increasing concentrations of HG (30,40, and 50 mM) for 24 h were incubated with the annexin V and PI and MitoSOX staining dye. The cell apoptosis and mitochondrial ROS were analyzed using flow cytometry and fluorescence microscopy. (d, e) WJMSCs were challenged with HG for 24 h, and the total cell lysate was harvested to analyze the expression of PTEN and downstream signaling cascade (AKT, p-AKT, FOXO3a, p-FOXO3a, and bim) via immunoblotting. (f) WJMSCs seeded in the presence of cycloheximide (CHX) were incubated with either MG-132 (10 μM) or HG (40 mM) for the indicated time (0, 3, 6, 9 h) and thereafter immunoblotted with the anti-PTEN antibody. β-actin served as a loading control. The scale bar indicates 50 μm. Values shown are means ± SD. Quantification of the results are shown (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 indicates the significant difference. HG, high glucose; PI, propidium iodide; PTEN, phosphatase and tensin homolog; ROS, reactive oxygen species; WJMSCs, Wharton's jelly derived mesenchymal stem cells of apoptotic cells (early, late) was significantly elevated in a dosedependent manner (Figure 1(b)). HG is the key regulator of mitochondrial ROS generation. Interestingly, immunofluorescence microscopy imaging showed increased ROS production with increasing HG concentrations ( Figure 1(c)). 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 HG-induced oxidative stress and apoptosis are regulated via the FOXO3a pathway. 37 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 (Figure 1(d),(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 inefficient degradation of PTEN (Figure 1(f)). The above data suggest that the PTEN/FOXO3a/Bim signaling pathway may be involved in HG-induced 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 (Figure 2(a)). Besides, other E3 ligases, including NEDD4-1, XIAP, and WWP2, that can ubiquitinate PTEN were analyzed F I G U R E 2 CHIP overexpressed WJMSCs attenuate hyperglycemia-induced PTEN mediated apoptosis and oxidative stress. (a) WJMSCs were challenged with increasing concentrations of HG for 24 h, and subsequently the expression level of the chaperone system (HSP70, HSP90, and CHIP) was measured using Western blotting. (b) WJMSCs were transfected with varying increasing concentration of CHIP (1, 2, and 3 μg) followed by HG incubation for 24 h. The cell viability was detected. (C, D) WJMSCs were transfected with HA-CHIP in the presence of HG (40 mM) for 24 h, and the mitochondrial ROS accumulation as well as apoptotic cell death were assessed using MitoSOX staining and flow cytometry. (e, f) WJMSCs were transfected with either HA-CHIP (3 μg) or siCHIP (30 nM) for 24 h in the presence of CHX (50 μg/ml) for indicated time points were subjected to HG challenge for 24 h, and the protein expression was measured using Western blot analysis. (g) WJMSCs were transfected with pRK5-HA-vector (3 μg), pRK5-HA-CHIP (3 μg), pRK5-HA-K30A (3 μg), and pRK5-HA-H260Q (3 μg), followed by HG incubation for 24 h. Cell lysates were immunoblotted to analyze the expression of PTEN and the downstream signaling cascade. β-actin act as a loading control. The scale bar indicates 100 μm. Values shown are means ± SD and quantification of the results shown as n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001 indicates the significant difference. CHIP, carboxyl terminus of Hsc70 interacting protein; HG, high glucose; PTEN, phosphatase and tensin homolog; ROS, reactive oxygen species; WJMSCs, Wharton's jelly derived mesenchymal stem cells using Western blotting and no observable difference was noticed in their expression levels under HG stress ( Figure S2(a)). Further, cell viability was retained dose dependently in CHIP overexpressing cells after HG induction (Figure 2(b)). 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 ( Figure S2(a)). 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 (Figure 2   Altogether, these data suggest 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
Above 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-immunoprecipitation (Co-IP) data showed that CHIP directly interacts with PTEN and promotes its ubiquitination (Figure 4(a)-(c)). These results indicate that F I G U R E 4 CHIP targets HG induced-PTEN for ubiquitin-mediated proteasomal degradation cooperated by HSP70 under HG conditions. (a-c) Cells transfected with HA-vector or HA-CHIP in the presence and absence of MG-132 for 6 h were subjected to HG challenge for 24 h. Whole cell lysate was immunoprecipitated with the anti-HA, anti-CHIP, and anti-PTEN antibody, and subsequently immunoblotted with the primary antibodies, including anti-HA, anti-PTEN, and anti-ubiquitin. (d, e) Cells transfected with HA-vector, HA-CHIP, and CHIP mutants (K30A, an H260Q) were treated with or without MG-132 for 6 h in the presence of HG for 24 h. Whole cell lysate was immunoprecipitated with the anti-HA and anti-PTEN antibody followed by immunoblotting with the anti-HA, anti-PTEN, and anti-ubiquitin antibody. (f) Cells were transfected with increasing concentrations of siHSP70 (10, 20, 30 nM) after challenged with HG for 24 h, and the expression level of PTEN and HSP70 was measured employing Western blot analysis. (g) Following cotransfection of GFP-vector or GFP-CHIP with increasing concentration of siHSP70 in WJMSCs were challenged with HG for 24 h, and the protein expression was measured via immunoblotting. (h) WJMSCs transfected with sicontrol, CHIP siRNA, or siHSP70 were subjected to HG challenge for 24 h, and the total cell extract was immunoblotted with CHIP, PTEN, and HSP70. β-actin served as a loading control. (i) Docking studies demonstrating the molecular interaction of HSP70 with PTEN forming a heteromer complex (HSP70 and PTEN shown in quaternary structure with helices and sheets in complex). Values shown are mean ± SD. Quantification of the results are shown (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 indicates the significant difference. CHIP, carboxyl terminus of Hsc70 interacting protein; HG, high glucose; PTEN, phosphatase and tensin homolog; WJMSCs, Wharton's jelly derived mesenchymal stem cells CHIP has the potential to interact with and promote ubiquitinmediated 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 (Figure 4(d),(e)). It is well known that CHIP regulates various proteins presented by the HSP70. Therefore, we evaluated the influence of HSP70 in CHIP-mediated PTEN degradation. We found that HSP70 inhibition led to a dose-dependent increase in PTEN protein expression (Figure 4 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. 2.5 | 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 influence 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 ( Figure 5(a)). Thereafter, we silenced FOXO3a together with AKT, and observed that AKT inhibition hindered elevation of the proapoptotic protein Bim (Figure 5(b)). We performed Western blotting to further ascertain the effect of CHIP on FOXO3a and its downstream promoter. Western blot analysis revealed that CHIP overexpression with shcontrol or siFOXO3a in the presence and absence of LY294002 (PI3K inhibitor) were subjected to HG for 24 h, and the activation of FOXO3a, p-FOXO3a, and Bim was analyzed using Western blot assay. (c) WJMSCs transfected with HA-vector, HA-CHIP, shcontrol, shCHIP, or siFOXO3a were incubated with HG for 24 h followed by immunoblotting to analyze the HA, FoxO3a, and Bim levels. (d) WJMSCs were transfected with varying concentration of siCHIP (10, 20, 30 nM) in the presence of HG for 24 h. Thereafter, the extracted cell lysates using cytoplasmic and nuclear fractionation kit were immunoblotted. (e) WJMSCs transfected with HA-CHIP or shCHIP plasmids were challenged with HG for 24 h and ChIP assay was performed to evaluate the FOXO3a interaction with the Bim promoter region. (f) Prediction of Bim promoter region using NCBI database (brown color indicates Bim promoter region) and converted to three-dimensional structure for molecular interaction with FOXO3a. (g) Docking studies illustrating the molecular interaction between FOXO3a and the Bim promoter region (Bim region is shown in green color and FOXO3a shown in red color). *p < 0.05, **p < 0.01, and ***p < 0.001 shows the significance. CHIP, carboxyl terminus of Hsc70 interacting protein; HG, high glucose; WJMSCs, Wharton's jelly derived mesenchymal stem cells immunoprecipitation (ChIP) assay was performed to analyze whether CHIP overexpression regulates the binding of FOXO3a with the Bim promoter region. It was revealed that HG increased the binding ability of FOXO3a with the Bim promoter fragment was reduced following CHIP overexpression; whereas, CHIP knockdown further increased their interaction ( Figure 5(e)). The active site of FOXO3a was predicted using the CASTp server, and includes amino acids TRP157, LEU160, LEU165, ARG168, CYS190, VAL190, and PRO192 ( Figure S5(b)). FOXO3a binds to the Bim promoter with high affinity and induces apoptosis. The Bim promoter region was selected from the NCBI database and drawn using Avogadro software ( Figure 5(f)).
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 files 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 ( Figure 5(g)).
2.6 | Coculture of CHIP overexpressing WJMSCs with cardiac cells ameliorates HG-induced cardiac apoptosis and oxidative stress However, CHIP overexpressed WJMSCs, and shPTEN expressing WJMSCs attenuated the blood glucose levels and AUC as well (Figure 7(b)). It was also noticed that no signs of tumorigenicity were observed during tissue sectioning of experimental animals after injecting WJMSCs carrying CHIP or shPTEN lentiviral plasmids. 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 significantly rescued the WHW and LVW weight in the experimental rats (Figure 7(c)).
Besides, LVW/WHW, WHW/tibia length (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-     39 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. 40 Ahmed et al. demonstrated that CHIP promotes the proteasomal degradation of PTEN. 41 In addition, CHIP has the ability to target many proteins for proteasomal degradation. 42,43 Our results are consistent with previous studies, wherein it has been highlighted that CHIP is able to promote the ubiquitination and proteasomal deg-

| Oral glucose tolerance test
After 6 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).

| Immunohistochemical staining
As mentioned above, the cardiac tissue sections were deparaffinized with xylene and rehydrated using graded series of alcohol followed by

| Reagents and antibodies
All chemicals and reagents were procured from Sigma Aldrich (Sigma

| Western blotting and immunoprecipitation
Western blot analysis was performed as described in our recent studies. 51 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 (1:3000 dilution) conjugated with HRP for 1 h at room temperature (RT). Finally, the analysis was obtained using enhanced chemiluminescence (ECL) kit (Millipore), and visualized with LAS 3000 imaging system (Fujifilm). All the images were quantified and analyzed using ImageJ (NIH) 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.

| 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) 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 min at 37 C, followed by 4 0 ,6diamidino-2-phenylindole (DAPI) for 5 min to examine the cell nuclei.
Mitochondrial ROS generation was measured using fluorescence microscopy (Olympus), with the excitation and emission wavelength in the range of 510/580 nm.

| TUNEL assay
After CHIP plasmid transfection and challenged with HG, cells were fixed with 4% Paraformaldehyde for 1 h at RT. 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). In cardiac tissue, slides were deparaffinized, 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 TUNELpositive cells using fluorescence microscopy (Olympus) 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.

| Chromatin immunoprecipitation
ChIP assay was performed to assess the probing of protein-DNA complex. SimpleChIP Enzymatic Chromatin IP kit (Magnetic beads, #9003) was procured from Cell Signaling Technology. ChIP assay was per-

| Coculturing
WJMSCs and H9c2 cardiomyocytes were cocultured in six-well Transwell inserts (Corning) with 0.4 μm pore size, and maintained in a 5% CO 2 humidified incubator. WJMSCs alone, WJMSCs expressing HA-CHIP, and HA-vector were seeded in the inner transwell chamber, while cardiac cells in the lower chamber were challenged with HG.

| Statistical analysis
Results are shown as mean ± SD. Statistical analysis was performed using GraphPad Prism5 statistical software. Multiple comparisons were accessed through one-way analysis of variance (ANOVA) and p value of <0.05 was considered statistically significant.