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, 28 (20), 6384-401

SIRT3 Is a Stress-Responsive Deacetylase in Cardiomyocytes That Protects Cells From Stress-Mediated Cell Death by Deacetylation of Ku70

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SIRT3 Is a Stress-Responsive Deacetylase in Cardiomyocytes That Protects Cells From Stress-Mediated Cell Death by Deacetylation of Ku70

Nagalingam R Sundaresan et al. Mol Cell Biol.

Abstract

There are seven SIRT isoforms in mammals, with diverse biological functions including gene regulation, metabolism, and apoptosis. Among them, SIRT3 is the only sirtuin whose increased expression has been shown to correlate with an extended life span in humans. In this study, we examined the role of SIRT3 in murine cardiomyocytes. We found that SIRT3 is a stress-responsive deacetylase and that its increased expression protects myocytes from genotoxic and oxidative stress-mediated cell death. We show that, like human SIRT3, mouse SIRT3 is expressed in two forms, a approximately 44-kDa long form and a approximately 28-kDa short form. Whereas the long form is localized in the mitochondria, nucleus, and cytoplasm, the short form is localized exclusively in the mitochondria of cardiomyocytes. During stress, SIRT3 levels are increased not only in mitochondria but also in the nuclei of cardiomyocytes. We also identified Ku70 as a new target of SIRT3. SIRT3 physically binds to Ku70 and deacetylates it, and this promotes interaction of Ku70 with the proapoptotic protein Bax. Thus, under stress conditions, increased expression of SIRT3 protects cardiomyocytes, in part by hindering the translocation of Bax to mitochondria. These studies underscore an essential role of SIRT3 in the survival of cardiomyocytes in stress situations.

Figures

FIG. 1.
FIG. 1.
Characterization of antibodies recognizing two forms of SIRT3 from cardiac tissue. (A) Schematic representation of hSIRT3 and mSIRT3 with epitope map of different antibodies used in this study. The asterisk indicates an antibody that was used only for immunostaining of cells. (B) Western blot analysis with SIRT3 antibody (AP6242a) showing two specific bands of SIRT3 in cardiomyocytes, as determined by using a blocking peptide. (C) SIRT3 levels in rat heart fibroblasts were knocked down by using SIRT3-specfic siRNA. Cell lysate was analyzed by Western analysis with the same antibody as that in panel B. Note the reduced levels of both forms of SIRT3 after knockdown of endogenous SIRT3 levels.
FIG. 2.
FIG. 2.
SIRT3 is a stress-responsive factor in cardiomyocytes. (A) Expression levels of SIRT3 in cardiomyocytes grown for 48 h in a medium with serum or defined medium without serum. Note induction of ∼44-kDa band but not 28-kDa band of SIRT3 in serum-free medium. (B) SIRT3 levels in cardiomyocytes treated with vehicle (Cont.) or MNNG at indicated concentrations for 24 h. (C) SIRT3 levels in cardiomyocytes treated with different concentrations of H2O2 for 24 h. (D and E) SIRT3 levels in cardiomyocytes treated with Ang-II (D) (5 μM) or PE (E) (20 μM) for 48 h. (F) Immunostaining of cardiomyocytes for ANF release from nuclei (white), a marker of cellular stress. Positions of nuclei were determined by DAPI staining. Values are means ± standard errors of three to four experiments. SIRT3 antibody AP6242a was used for Western analysis.
FIG. 3.
FIG. 3.
SIRT3 overexpression protects cardiomyocytes from genotoxic and oxidative stress-mediated cell death. (A) Expression levels of hSIRT3 in cardiomyocytes infected with adenovirus (Ad) vector for 24 h were determined by Western analysis with SIRT3 antibody PAB-11098. Note the presence of both forms of hSIRT3 in adenovirus-infected cells. (B) Deacetylase activity of hSIRT3 in cardiomyocytes. Cells infected with viral vectors, synthesizing the wt SIRT3 or an mt (benign) protein, were treated with PE to induce cellular acetylation. Acetylation of histones was determined by Western analysis. (C) Quantification of H3 deacetylation in SIRT3-overexpressing cells. (D to F) Cardiomyocytes overexpressing hSIRT3 or the mt protein were treated with MNNG (500 μM), camptothecin (Camp.; 10 μM), or H2O2 (200 μM). Cell death was determined 6 h after treatment by Hoechst and PI staining followed by FACS analysis. (G) Western analysis of the cell lysate with a poly(ADP-ribose) antibody, indicating PARP1 activity. Quantitative values are means ± standard errors of five to seven plates of three separate experiments.
FIG. 4.
FIG. 4.
SIRT3 expression is required for survival of HeLa cells after genotoxic stress. (A) Representative Western blot showing endogenous expression of SIRT3 in control cells and cells subjected to SIRT3 knockout. Note reduced levels of both forms of SIRT3 after siRNA-mediated knockdown. (B) Western blot showing expression of SIRT3 in control and stable HeLa cells. (C) Western blot showing reduced levels of both forms of SIRT3 after siRNA-mediated knockdown in stable cells. (D) HeLa cells (control and endogenous SIRT3 knockdown cells) were treated with MNNG (500 μM) for 10 min and then washed and kept in complete medium for 6 h. Cell death was determined by Hoechst and PI staining followed by FACS analysis. (E) SIRT3 stable cells expressing wt SIRT3 or mt SIRT3 were examined for MNNG-mediated cell death. (F) SIRT3 stable cells were subjected to SIRT3 knockdown by siRNA and then tested for MNNG-mediated cell death. SIRT3 antibody PAB-11098 was used in all these Western blot assays. Values are means ± standard errors of three experiments.
FIG. 5.
FIG. 5.
SIRT3 is localized in the cytoplasm and mitochondria as well as nuclei of heart tissue. (A) Nuclear (Nucl.) and nonnuclear (Non Nucl.) fractions of mouse heart were prepared by using the Pierce cell fractionation kit. Fractions were characterized by Western blotting taking GAPDH and MnSOD as nonnuclear markers and RNA polymerase II as a nuclear marker. Both fractions were analyzed for the expression level of SIRT3 by Western blotting. Actin was utilized as a loading control. Note the presence of the (∼44-kDa) long form of SIRT3 in both the nuclear and nonnuclear fractions, while the ∼28-kDa short form is detected only in the nonnuclear fraction. (B) Three subcellular fractions (cytoplasmic, mitochondrial, and nuclear) of a mouse heart were prepared, utilizing the Calbiochem fractionation kit, and characterized by Western blotting using fraction-specific protein antibodies. All three fractions were also analyzed for expression of mSIRT3 by Western blotting. The SIRT3 antibody Ab56214 was used in these blotting assays. Note the presence of ∼44-kDa SIRT3 in all three fractions, whereas the short form (∼28 kDa) of SIRT3 was detected only in the mitochondrial fraction of the heart.
FIG. 6.
FIG. 6.
Microscopic analysis of heart tissue showing nuclear localization of mSIRT3. (A, B, and D) Representative electron micrographs of mouse heart sections stained with anti-SIRT3 antibody (PAB-11098). Panel B is an enlarged portion of the nucleus shown in panel A. (C and E) Heart sections stained with nonspecific IgG. N, nucleus; Mit, mitochondria; Myo, myofilaments. Numbers at the top of the picture indicate magnifications. High-density black dots (purple arrows) indicate the localization of SIRT3 in the nucleus and mitochondria. (F) Confocal microscopic image of a cardiomyocyte stained with anti-SIRT3 antibody (SC49744) (red). Positions of nuclei and mitochondria were detected by DAPI (blue) and Mitotracker (green) staining, respectively. Note the presence of SIRT3 (red in the merged picture) in the cytoplasm and mitochondria as well as the nuclei of cardiomyocytes.
FIG. 7.
FIG. 7.
Overexpressed SIRT3 is localized in the cytoplasm and mitochondria as well as the nuclei of cardiomyocytes. Representative confocal microscopic images of cardiomyocytes infected with ad.hSIRT3 vector and stained with anti-SIRT3 antibody (SC49744) (red). Mitotracker and DAPI staining was utilized for localization of mitochondria and nuclei, respectively. (A) Control cells treated with vehicle. (B) Cells treated with Ang-II (5 μM) for 48 h. Note the induction of SIRT3 in both the mitochondria and nuclei of the cell. (C) Negative control in which a specific blocking peptide was used to validate the specificity of SIRT3 antibody. (D) Cells were overexpressed with ad.hSIRT3 and treated with vehicle or MNNG (500 μM) for 6 h. Subcellular fractions (nuclear and nonnuclear) were prepared and analyzed for the expression of SIRT3 by Western analysis (SIRT3 antibody PAB-11098). GAPDH was utilized as a cytosolic marker, and tubulin was used as a loading control. (E) Quantification of SIRT3 induction after MNNG treatment of cells. Note induction of both the 44- and 28-kDa forms of SIRT3 after genotoxic stress. (F) SIRT3Δ1-25 deletion mt lacking the mitochondrial import signal is effective in protecting cells from MNNG-mediated cell death. HeLa cells were overexpressed with wt SIRT3, mt SIRT3 (catalytically inert), or SIRT3Δ1-25. Cells were treated with MNNG (500 μM), and cell death was monitored 3 h later. Note significantly reduced cell death in SIRT3Δ1-25-expressing cells compared to that of cells overexpressing catalytically inert protein. Cont, control. Values are means ± standard errors of four separate experiments. (G) Localization of SIRT3Δ1-25 deletion mt in the nucleus but not in the nonnuclear fraction.
FIG. 8.
FIG. 8.
SIRT3 interacts with Ku70 in vitro and in vivo. (A) Endogenous SIRT3 interacts with Ku70. Cardiomyocyte lysate was subjected to IP with either nonspecific IgG or specific anti-SIRT3 antibody (AP6242a). Resulting beads were analyzed by Western blotting (WB) with anti-Ku70 antibody. (B) Cos7 cells were induced to overexpress with the Flag tag or the Flag-tagged SIRT3. Cell lysate was subjected to IP with either nonspecific IgG-conjugated beads or anti-Flag M2 agarose beads. Precipitated beads were analyzed by Western blotting with anti-Ku70 antibody. (C) Cells infected with ad.hSIRT3 vector were subjected to IP with Ku70 antibody, and the resulting beads were analyzed by Western blotting with anti-SIRT3 antibody (PAB11098). Note the presence of both forms of SIRT3 in the input lane, but only the long form of SIRT3 was pulled down by Ku70 in this assay. (D) In vitro protein binding assay. In vitro-synthesized [35S]methionine-labeled Ku70 was incubated with beads containing His-tagged 28-kDa SIRT3, His-tagged p38d, or nickel beads alone. His-tagged proteins were precipitated as nickel resin beads, and bound proteins were analyzed by SDS-PAGE. (E) Picture of a Coomassie blue-stained gel showing synthesis of the His-tagged 28-kDa form of SIRT3 from plasmid (His-SIRT3119-398).
FIG. 9.
FIG. 9.
SIRT3 deacetylates Ku70 under in vitro and in vivo assay conditions. (A) In an acetylation buffer Flag.Ku70 was incubated with PCAF. Acetylation of protein was determined by Western blotting with antiacetyllysine antibody. (B) Deacetylation of Ku70 by SIRT3 in vitro. Flag.Ku70 was acetylated in vitro with PACF, and it was precipitated with Flag M2 beads. Acetylated Flag.Ku70 was then incubated with beads containing wt or mt SIRT3 in a deacetylation buffer with or without NAD. Both wt and mt SIRT3 were immunoprecipitated from stable HeLa cells. (C) Quantification of Ku70 deacetylation by SIRT3. (D) In vivo deacetylation of Ku70 by SIRT3. Stable cells expressing wt or mt SIRT3 were induced to overexpress with Flag.Ku70 and treated with NAM (10 mM for 24 h) and/or TSA (5 μM for 6 h) as indicated. Flag.Ku70 was immunoprecipitated, and the level of acetylation was analyzed by probing with antiacetyllysine antibody. (E) Quantification of Ku70 deacetylation by SIRT3 in vivo. Values are means of three experiments.
FIG. 10.
FIG. 10.
SIRT3 blocks Ku70 acetylation during stress. (A) Ku70 was acetylated during genotoxic and oxidative stress. Cells were transiently expressed with Flag-Ku70 and treated with MNNG (500 μM) or H2O2 (500 μM). Cells were harvested at indicated time intervals after treatment, and Flag.Ku70 was immunoprecipitated. Flag.Ku70 beads were analyzed by Western blotting using antiacetyllysine and anti-Ku70 antibodies. (B) Quantitative representation of Ku70 acetylation by MNNG and H2O2 treatment of cells. Values are means of three separate experiments. (C) HeLa cells stably expressing either wt or mt SIRT3 were treated with MNNG for 2 h. The endogenous Ku70 was immunoprecipitated and analyzed by Western blotting as in panel A. (D) Knockdown of SIRT3 increases Ku70 acetylation during genotoxic stress. SIRT3 was knocked down from SIRT3-expressing stable cells by use of SIRT3-specific siRNA. Scrambled RNA was used as a negative control. Cells were treated with MNNG for 2 h, and the level of Ku70 acetylation was determined by Western blotting.
FIG. 11.
FIG. 11.
SIRT3 prevents Bax-mediated apoptosis by deacetylating Ku70. (A) HeLa cells stably expressing wt SIRT3 (black bars) or mt SIRT3 (white bars) were transfected with plasmids synthesizing YFP, YFP.Bax, or YFP.Bax and Ku70 together. The percentage of YFP-positive cells (yellow fluorescence) with apoptotic (fragmented) nuclei was scored 12 h after transfection. Values are the averages of three experiments; during each experiment >200 cells were scored. (B) HeLa cells stably expressing wt SIRT3 were given SIRT3-specific siRNA or scrambled RNA. We observed >80% reduction of the SIRT3 levels in cells to which siRNA was added, as shown in Fig. 4C. These cells were then transfected with plasmids encoding YFP.Bax and Ku70. Cell death was scored 12 h posttransfection. Values are the averages of four experiments. (C) Salvage of wt SIRT3 levels protects cells from Bax-mediated cell death. SIRT3-knockdown HeLa cells were infected with ad.hSIRT3 or the mt vector. After 12 h of virus infection, cells were transfected with YFP.Bax and Ku70 plasmids together. The percentage of YFP-positive cells with apoptotic nuclei was scored 12 h after transfection. Values are the means of four experiments with >200 cells scored in each plate. Nearly 80% of SIRT3 levels were recovered in these cells, as verified by Western blotting (not shown). (D) Representative picture of HeLa cells subjected to Bax-mediated apoptosis. Cells were infected with ad.hSIRT3 or the mt (ad.mt) vector. Twenty-four hours after viral infection, cells were transfected with different plasmids as indicated. The pictures of cells were taken 12 h posttransfection. Note the presence of fragmented apoptotic nuclei in cells transfected with YFP.Bax and YFP.Bax plus Ku70 (middle panels) but not in SIRT3-overexpressing cells (bottom panels). (E) SIRT3 blocks localization of Bax to mitochondria during genotoxic stress. Cardiomyocytes infected with ad.hSIRT3 or the mt (ad.mt) vectors were treated with MNNG for 6 h. Cells were stained with anti-Bax antibody (red). DAPI (blue) staining and Mitotracker (green) staining were utilized as markers of nuclei and mitochondria, respectively. Note shrunken nuclei in upper panels (ad.mt) depicting cell death. In these cells, Bax was totally merged with Mitotracker green (yellow in merged image) representing mitochondrial localization of Bax. In the lower panels, cells overexpressing wt SIRT3 and having well-preserved nuclei showed no Bax translocation to mitochondria (no yellow in merged image).
FIG. 12.
FIG. 12.
Redundant function of SIRT3 and SIRT1 of protecting cells from apoptosis. (A) SIRT3- and SIRT1-mediated deacetylation of Ku70 enhances its binding to Bax. Flag-Ku70 was synthesized in vitro, captured on Flag beads, and then acetylated by PCAF. Acetylated Flag.Ku70 was subjected to deacetylation by SIRT1, SIRT3, or the mt protein. The acetylated and deacetylated Flag.Ku70 was then incubated with HeLa cell extract in CHAPS buffer overnight. Flag.Ku70 beads were separated from the extract and tested for Bax coprecipitation. Beads were also tested for the acetylation status of Ku70 by Western blotting using appropriate antibodies. Note that Bax was pulled down by Ku70 that was deacetylated by either SIRT1 or SIRT3 but not by the acetylated Ku70. (B) SIRT1 was knocked out (KO) from wt SIRT3- or mt SIRT3-expressing stable cell lines, using SIRT1-specific siRNA. The blot shows Western analysis of SIRT1 expression levels in control and KO cells. (C) SIRT1 KO sensitized mt SIRT3-expressing cells, but not the wt SIRT3-expressing cells, to MNNG-mediated cell death. Values are means ± standard errors of four experiments. (D) Representative density blots of FACS analysis of cells treated with MNNG (500 μM). Cell death was monitored 3 h later.

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