Sirtuin3 protects aged human mesenchymal stem cells against oxidative stress and enhances efficacy of cell therapy for ischaemic heart diseases

Abstract Sirtuin3 (SIRT3) is associated with oxidative stress and lifespan. However, the possible mechanisms underlying its influence are unknown. We hypothesized that SIRT3 increases the antioxidant capacity of aged cells and improves the efficacy of human mesenchymal stem cell (hMSC) therapy for ischaemic heart diseases in aged patients. In vitro, the antioxidant capacity of old hMSCs (O‐hMSCs) was increased after SIRT3 overexpression using a gene transfection technique, while the antioxidant capacity of young hMSCs (Y‐hMSCs) was decreased by SIRT3 silencing. The levels of forkhead box O3a (FoxO3a) in the nucleus, and antioxidant enzymes Mn‐superoxide dismutase (MnSOD) and catalase (CAT) increased in SIRT3‐overexpressed O‐hMSCs while they decreased in SIRT3‐silenced Y‐hMSCs after oxidative stress. Following myocardial infarction in adult rats in vivo, infarct size decreased and cardiac function was significantly enhanced after cell transplantation with SIRT3 overexpressed O‐hMSCs. The number of apoptotic cells decreased and the survival rate of transplanted cells increased following SIRT3 overexpression in O‐hMSCs. SIRT3 protects aged hMSCs against oxidative stress by positively regulating antioxidant enzymes (MnSOD and CAT) via increasing the expression of FoxO3a in the nucleus. The efficacy of aged hMSC transplantation therapy for ischaemic heart diseases can be improved by SIRT3 overexpression.

therapy for ischaemic heart diseases. [4][5][6] In early animal experiments, the treatment effect of young stem cell transplantation was satisfactory. 5 However, the therapeutic effect from old autologous stem cell transplantation was found to be limited in clinical trials. 7,8 Our previous studies have revealed the close association between stem cell function and ageing. 9,10 The regenerative capacity, biological activity and resistance to oxidative stress of stem cells significantly decline with age. This suggests that stem cell senescence is likely to be a major contributing factor to the ineffectual cell transplantation outcomes found in the clinical setting. We hypothesized that enhancing the function of old stem cells may improve the therapeutic effect of transplantation.
Sirtuin3 (SIRT3) is a deacetylase from the sirtuin family. 11,12 There is significant evidence that SIRT3 localizes to mitochondria and plays a positive role in human longevity, organ ageing and stem cell function. [13][14][15] Human SIRT3 has two isoforms: a full-length isoform (fl-SIRT3) and a short one (sh-SIRT3) which is considered to have the most functional significance. [16][17][18][19] Recent studies have reported that SIRT3 reduces levels of reactive oxygen species (ROS) by deacetylating the transcription factor forkhead box O3a (FoxO3a), which can then enter the nucleus and bind to the promoter of the genes encoding Mn-superoxide dismutase (MnSOD) and CAT. [20][21][22] Furthermore, many researchers have demonstrated that the elevated expression of MnSOD and CAT by SIRT3 overexpression can protect cells against the oxidative damage induced by ROS. 10,17 In our previous study, we determined that the lower expression of sh-SIRT3 in old relative to young human myocardial tissue significantly contributed to myocardial ageing. 10 In addition, we showed that the decline in antioxidant capacity of old human MSCs (O-hMSCs) is because of a substantially greater down-regulation of SIRT3 than that which occurs in young hMSCs (Y-hMSCs) under oxidative stress. 10 However, there is little evidence to support the potential association between SIRT3 and O-hMSC function. In this study, we explored the relationship and underlying mechanisms involved in SIRT3's influence on the antioxidant capacity of O-hMSCs. We further investigated the efficacy of cell transplantation therapy when SIRT3-modified O-hMSCs were injected into the infarct region of the heart using a rat myocardial infarction (MI) model. Helsinki. O-hMSCs were obtained from patients 50-72 years of age (mean = 61.3 ± 9.5), and Y-hMSCs were obtained from patients 0-12 years of age (mean = 5.0 ± 4.9). MSCs were isolated by density gradient separation with Ficoll-Paque premium (1.073 g/mL density; GE Healthcare, Uppsala, Sweden) as described previously. 10

| Plasmid and siRNA constructs
A plasmid containing a SIRT3 expression gene (pSIRT3) was constructed on a pEX-1 (pGCMV/MCS/EGFP/NEO) backbone (Gene-Pharma, Shanghai, China). The plasmids were amplified and prepared using the Endofree maxi plasmid kit (TIANGEN, Beijing, China). The negative control (NC) and small interfering RNA (siRNA) were designed and synthesized by GenePharma to interfere with SIRT3 expression.

| Plasmid and siRNA transfection
The plasmid transfection was performed in accordance with the manufacturer's instructions (X-tremeGENE HP DNA transfection reagent; Roche, Penzberg, Upper Bavaria, Germany). After 72-hour culture, transfection efficiency was assayed by fluorescence activated cell sorting (FACS; BD, Franklin Lakes, NJ, USA). The siRNA transfection was performed in accordance with the manufacturer's instructions (X-tremeGENE HP siRNA transfection reagent; Roche).

| Cell stress experiment
The transfection was performed as described above.

| Gene expression measurement
Total RNA was extracted directly from hMSCs using TRIzol reagent

| Enzyme activity measurement of MnSOD and CAT
To measure enzyme activity, cultured cells were collected and protein extracted as outlined above. Enzyme activity was detected using the MnSOD Assay and CAT Assay kits (Beyotime) according to the manufacturer's protocol.

| Infarct size measurement
Four weeks after MI and cell transplantation, rats were anaesthetized and respiration was assisted as described above. The heart was exposed through a median sternotomy, and 0.9% normal saline was continuously injected into the left ventricle of rats through the apex cordis using a syringe (20 mL

| Cardiac function assessment
Prior to and 1, 2 and 4 weeks after MI, rats were anaesthetized as

| Cell apoptosis and survival evaluation in vivo
Three days after MI and cell transplantation, rats were sacrificed.
Briefly, rats were anaesthetized and respiration was assisted as above. The heart was exposed through a median sternotomy, the right atrium was carved to reduce the circulation load and 0.9% normal saline was continuously injected into the left ventricle through the apex cordis using a syringe (20 mL) until cardiac arrest occurred.
The hearts were excised as described above and sliced into 2-mm thick sections and fixed with 4% paraformaldehyde for 24 hours, followed by sucrose at varying concentrations and durations (10% for 1 hour, 20% for 1 hour and 30% for 24 hours). The tissues were then snap-frozen in moulds filled with optimal cutting temperature (OCT) compound (SAKURA, Torrance, CA, USA). The OCT-embedded tissues were cut into 5-μm-thick sections using a freezing microtome.

| Statistical analysis
Data are expressed as mean ± standard deviation (SD). Analyses were conducted using GraphPad Prism 6.0 software (GraphPad, La Jolla, CA, USA). Comparisons between two groups were performed using two-tailed Student's t test. One-way ANOVA was used to determine the significance between three or more experimental groups. Repeated-measures ANOVA was used for left ventricular systolic function (ejection fraction and fractional shortening).
P < 0.05 was considered statistically significant.

| Expression of SIRT3, MnSOD and CAT increase after transfection of SIRT3 in O-hMSCs
To study the effect of SIRT3 on the function of O-hMSCs, SIRT3 was transfected into O-hMSCs. Flow cytometry data showed that transfection efficiency was 14.1 ± 1.7% ( Figure 1A,B, n = 6/group).
The gene and total protein expression of SIRT3 in the SIRT3 group was significantly higher compared to the Control group ( Figure 1C,D, n = 6/group). Moreover, the protein expression of fl-SIRT3 and sh-SIRT3 both increased in the SIRT3 group compared with the Control group ( Figure 1E, n = 6/group). To further verify the activity of SIRT3 after transfection, gene and protein expression as well as activity of MnSOD and CAT were found to be increased after SIRT3 overexpression ( Figure 1F-K, n = 6/group). These results demonstrate that SIRT3 was successfully overexpressed in O-hMSCs using plasmid transfection, which correlated with antioxidant activity.

| Enhanced antioxidant capacity in O-hMSCs protects cells from oxidative stress injury
As shown in Figure 2, the rate of apoptosis increased while the rate of cell survival decreased after oxidative stress induced by H 2 O 2 .
The apoptosis rate was significantly lower and the survival rate significantly higher in the SIRT3+ group than the Control+ group, while there were no significant differences between the SIRT3 and Control groups, which were free from oxidative stress (n = 6/group). We fur- and SIRT3+ groups than in the non-H 2 O 2 -treated groups (Control and SIRT3). However, these levels were still significantly higher in the SIRT3+ group than the Control+ group ( Figure 3A-C). The mRNA, protein and enzyme activity levels of MnSOD and CAT which decreased after oxidative stress were still significantly higher in the SIRT3+ group than the Control+ group ( Figure 3D-I). H,K, Enzyme activity of MnSOD and CAT was significantly greater in the SIRT3 group compared with the Control group (two-tailed t test; *P < 0.05; **P < 0.01; n = 6/group) After pSIRT3 transfection, FoxO3a protein expression significantly increased in the nuclear fraction ( Figure 3J). After H 2 O 2 treatment, the expression of FoxO3a protein in the nucleus was significantly lower in the Control+ and SIRT3+ groups than the Control and SIRT3 groups. However, the level was still significantly higher in the SIRT3+ group than the Control+ group ( Figure 3J).
These findings suggest that the antioxidant capacity of O-hMSCs was enhanced by SIRT3 overexpression, likely by increasing expression and activity of MnSOD and CAT via transferring FoxO3a into the nucleus.

| Silencing SIRT3 reduces antioxidant capacity of Y-hMSCs
The expression of SIRT3 was reduced using silencing RNA transfection in Y-hMSCs. The rate of apoptosis increased while the cell survival rate decreased after cells were exposed to H 2 O 2 treatment.
Cellular apoptosis was significantly higher and cell survival significantly lower in the siSIRT3+ group than the Control+ group, and there were no differences between the Control and siSIRT3 groups (without H 2 O 2 treatment; Figure

| Cardiac function is enhanced following transplantation of SIRT3-overexpressed O-hMSCs
Echocardiography was conducted before and 1, 2 and 4 weeks after

| DISCUSSION
The efficacy of current therapies for ischaemic heart diseases in aged patients is limited. 2,3 In previous pre-clinical studies, young MSC transplantation has been demonstrated as an effective therapy for ischaemic heart diseases. 5 The use of hMSCs is safe and feasible in the clinical setting, and these cells have been used in multiple clinical trials. 8 However, most studies used autologous O-hMSCs because most patients with ischaemic heart diseases are older. 7,23 The efficacy of these aged cells was found to be unsatisfactory potentially because of their decreased function. 9 Improving the function of O-hMSCs may improve the therapeutic effect of autologous stem cell transplantation in older patients.
Sirtuin3 (SIRT3) is a class III deacetylase depending on the nicotinamide adenine dinucleotide (NAD+) and belongs to the highly conserved Sirtuin family. 11,12,19 As a mitochondria-localized protein, SIRT3 mainly exists in tissues and organs that are rich in mitochondria such as the kidney, brain, heart and liver. 18 Human SIRT3 has two isoforms: fl-SIRT3 and sh-SIRT3. [16][17][18][19] Previous studies, including our own, have shown that the short isoform of SIRT3 has the most functional significance. [16][17][18][19] In the current study, the expression level of sh-SIRT3 was down-regulated by oxidative stress which is consistent with previous research. [16][17][18] SIRT3 has been shown to be protective against oxidative stress, but the majority of these studies were performed on animals, cell lines and tumour cells. 17

ACKNOWLEDG EMENTS
We thank Dr. Leigh Botly for help with manuscript preparation and editing. This work was supported by grants from the National Natural Science Foundation of China (81270188, 81401203, 81770347, and 81500268) and the Natural Science Foundation of Heilongjiang Province of China (JC2015020, LC2015040).

CONFLI CT OF INTEREST STATEMENT
The authors confirm that there are no conflicts of interest. F I G U R E 6 Infarct size was decreased and cardiac function was enhanced after cell transplantation with old human mesenchymal stem cells (hMSCs) modified by sirtuin3 (SIRT3). A, Masson's Trichrome staining of the infarct size 4 wk after cell transplantation in the three groups (blue = collagen; red = myocardium). Scale bars represent 2 mm. B, The infarct size of the O-hMSCs-pSIRT3 group was significantly smaller than the medium control and O-hMSCs-pEX-1 groups 4 wk after myocardial infarction (MI), and there was no significant difference between the medium control and pEX-1 groups. C, Representative echocardiography images from the three groups before and 1, 2 and 4 wk after MI (solid lines: left ventricular end-systolic diameter; dashed lines: left ventricular end-diastolic diameter). D,E, The ejection fraction (EF) and fractional shortening (FS) of the O-hMSCs-pSIRT3 group after MI were significantly higher than the medium control and O-hMSCs-pEX-1 groups, and there was no significant difference between the medium control and O-hMSCs-pEX-1 groups. F,G, The left ventricular end diastolic volume (LVEDV) and left ventricular end systolic volume (LVESV) of the O-hMSCs-pSIRT3 group after MI were significantly smaller than that of the medium control and O-hMSCs-pEX-1 groups, and there were no significant differences between the medium control and O-hMSCs-pEX-1 groups. (ANOVA; **P < 0.01; n = 6/group)