Decreased SIRT3 in aged human mesenchymal stromal/stem cells increases cellular susceptibility to oxidative stress

Abstract

Sirtuin3 (SIRT3) is an important member of the sirtuin family of protein deacetylases that is localized to mitochondria and linked to lifespan extension in organisms ranging from yeast to humans. As aged cells have less regenerative capacity and are more susceptible to oxidative stress, we investigated the effect of ageing on SIRT3 levels and its correlation with antioxidant enzyme activities. Here, we show that severe oxidative stress reduces SIRT3 levels in young human mesenchymal stromal/stem cells (hMSCs). Overexpression of SIRT3 improved hMSCs resistance to the detrimental effects of oxidative stress. By activating manganese superoxide dismutase (MnSOD) and catalase (CAT), SIRT3 protects hMSCs from apoptosis under stress. SIRT3 expression, levels of MnSOD and CAT, as well as cell survival showed little difference in old versus young hMSCs under normal growth conditions, whereas older cells had a significantly reduced capacity to withstand oxidative stress compared to their younger counterparts. Expression of the short 28 kD SIRT3 isoform was higher, while the long 44 kD isoform expression was lower in young myocardial tissues compared with older ones. These results suggest that the active short isoform of SIRT3 protects hMSCs from oxidative injury by increasing the expression and activity of antioxidant enzymes. The expression of this short isoform decreases in cardiac tissue during ageing, leading to a reduced capacity for the heart to withstand oxidative stress.

Keywords: SIRT3; ageing; antioxidants; mesenchymal stromal/stem cells; myocardium; oxidative stress.

Figure 1

Oxidative stress leads to decreased SIRT3, CAT and MnSOD levels in young hMSCs correlated with decreased cell survival and increased apoptosis. (A) hMSCs was treated with 1 mM H₂O₂ for 1 hr and gene expression was evaluated by real-time PCR. SIRT3 mRNA expression was reduced in young hMSCs under oxidative stress. (B and C) show total SIRT3 protein expression levels as well as the expression levels of its two isoforms (full-length SIRT3, fl-SIRT3 and short SIRT3, sh-SIRT3) before and after H₂O₂ treatment, assessed by Western blot with β-actin serving as an internal control. (D and G) show MnSOD and CAT gene expression before and after treatment. (E and H) MnSOD and CAT protein expression assayed by Western blot. (F and I) Enzymatic activity of MnSOD and CAT in control and H₂O₂-treated groups. (J) Cell survival evaluated by Cell Counting Kit-8 (CCK8). (K and L) Cell apoptosis assessed by flow cytometry and 4′,6-diamidino-2-phenylindole (DAPI) staining in control and H₂O₂-treated groups. Red arrows indicate apoptotic cells and some cells are enlarged to show the stages of apoptotic degradation: the nuclear membrane edge of phase I apoptotic cells is rippled (left panel, insets), while the chromatin of phase II apoptotic is highly condensed and marginalized (right panel, red arrows and inset), and finally apoptotic bodies are formed.

Figure 2

Transfection of pSIRT3 enhances SIRT3, CAT and MnSOD expression in young hMSCs. (A) Transfection efficiency of young hMSCs (23.6% ± 3.1%, n = 3) was detected by fluorescence-activated cell sorting. (B and C) showed SIRT3 gene and protein expression in the pEX-1- and pSIRT3-transfected groups. (D) It shows full-length (fl-SIRT3) and short SIRT3 (sh-SIRT3) expression levels in the pEX-1- and pSIRT3-transfected groups. (E–I) Gene and protein levels of MnSOD and CAT were increased after pSIRT3 transfection compared with the pEX-1-transfected group. (G and J) Enzymatic activity of MnSOD and CAT were also enhanced after pSIRT3 transfection compared with the pEX-1-transfected group.

Figure 3

SIRT3 overexpression enhances the ability of young hMSCs to protect against oxidative stress. (A and B) SIRT3 gene and protein expression in pEX1 and pSIRT3 transfection groups with and without H₂O₂ treatment. (C) Full-length and sh-SIRT3 expression in pEX-1- and pSIRT3-transfected cells with or without oxidative stress. (D–H) Gene and protein levels of MnSOD and CAT are higher in the pSIRT3 group than the pEX1 group with and without H₂O₂ treatment. (F and I) The activity of MnSOD and CAT in pEX-1 and pSIRT3 groups with and without H₂O₂ treatment. (J and K) Cell survival and cell apoptosis in pEX-1 and pSIRT3 groups with or without H₂O₂ treatment. Overall, MnSOD and CAT expressions, function and cell survival are increased, while cell death is decreased in cells transfected with pSIRT3 than those transfected with pEX-1. Oxidative stress reduces these effects, but cells transfected with the SIRT3 expression construct are still protected against H₂O₂-induced cell death and show increased antioxidant expression and function in the presence of H₂O₂ than cells expressing empty plasmid.

Figure 4

SIRT3, MnSOD and CAT expression in young and old hMSCs and myocardial tissues. (A and B) There were no differences in SIRT3 gene and protein expression between young (Y) and old (O) hMSCs in normal culture conditions. (C) The protein expression of full-length (fl-SIRT3) and the short isoform of SIRT3 (sh-SIRT3) in young and old hMSCs. (D–H) There were no differences in MnSOD and CAT gene and protein expression between young and old hMSCs. (F and I) The enzymatic activity of MnSOD and CAT in the young and old cell groups. (J–L) Cell survival and apoptosis in young cells compared with old cells. No differences in cell survival or apoptosis were seen in the absence of cellular stress. (M and N) mRNA and total protein expression of SIRT3 in young and old myocardial tissues. (O) fl-SIRT3 and sh-SIRT3 expression in young and old myocardial tissues. Despite there being no difference in SIRT3 gene expression, sh-SIRT3 is found at a significantly higher level in young compared to old cells, while fl-SIRT3 is decreased in these cells. (P–T) MnSOD and CAT gene and protein levels in young and old cell groups. (R and U) Both MnSOD and CAT activities are decreased in old myocardial tissue.

Figure 5

The antioxidant ability of old hMSCs was significantly decreased compared with that of young cells under oxidative stress. (A and B) SIRT3 mRNA and total SIRT3 protein expression in young and old hMSCs under oxidative stress. SIRT3 expression is significantly decreased in old cells compared with their younger counterparts. (C) The aged hMSCs expressed lower levels of full-length SIRT3 (fl-SIRT3) and the short isoform of SIRT3 (sh-SIRT3) compared with young cells after H₂O₂ treatment. (D–H) mRNA and protein levels of MnSOD and CAT are reduced in older cells treated with H₂O₂. (F and I) MnSOD and CAT enzymatic activity in old hMSCs was significantly lower than that in young ones under the same oxidative stress. (J–L) Cell survival and apoptosis of young and old groups after H₂O₂ treatment. Older cells show increased apoptosis and reduced cell survival under conditions of oxidative stress than do their younger counterparts.