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Mild endothelial dysfunction in Sirt3 knockout mice fed a high-cholesterol diet: protective role of a novel C/EBP-β-dependent feedback regulation of SOD2.

Winnik S, Gaul DS, Siciliani G, Lohmann C, Pasterk L, Calatayud N, Weber J, Eriksson U, Auwerx J, van Tits LJ, Lüscher TF, Matter CM - Basic Res. Cardiol. (2016)

Bottom Line: Sirtuin 3 (Sirt3) is an NAD(+)-dependent mitochondrial deacetylase associated with superoxide dismutase 2 (SOD2)-mediated protection from oxidative stress.Thus, we aimed to unravel the effects of endogenous Sirt3 on endothelial function and oxidative stress.In cultured endothelial cells, a novel C/EBP-β-dependent rescue mechanism maintains net SOD2 activity upon transient knockdown of Sirt3.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, University Heart Center Zurich, University Hospital Zurich, Raemistr. 100, 8091, Zurich, Switzerland. stephan.winnik@usz.ch.

ABSTRACT
Sirtuin 3 (Sirt3) is an NAD(+)-dependent mitochondrial deacetylase associated with superoxide dismutase 2 (SOD2)-mediated protection from oxidative stress. We have reported accelerated weight gain and impaired metabolic flexibility in atherosclerotic Sirt3 (-/-) mice. Oxidative stress is a hallmark of endothelial dysfunction. Yet, the role of Sirt3 in this context remains unknown. Thus, we aimed to unravel the effects of endogenous Sirt3 on endothelial function and oxidative stress. Knockdown of Sirt3 in human aortic endothelial cells (HAEC) increased intracellular mitochondrial superoxide accumulation, as assessed by electron spin resonance spectroscopy and fluorescence imaging. Endothelium-dependent relaxation of aortic rings from Sirt3 (-/-) mice exposed to a normal diet did not differ from wild-type controls. However, following 12 weeks of high-cholesterol diet and increasing oxidative stress, endothelial function of Sirt3 (-/-) mice was mildly impaired compared with wild-type controls. Relaxation was restored upon enhanced superoxide scavenging using pegylated superoxide dismutase. Knockdown of Sirt3 in cultured HAEC diminished SOD2 specific activity, which was compensated for by a CCAAT/enhancer binding protein beta (C/EBP-β)-dependent transcriptional induction of SOD2. Abrogation of this feedback regulation by simultaneous knockdown of C/EBP-β and Sirt3 exacerbated mitochondrial superoxide accumulation and culminated into endothelial cell death upon prolonged culture. Taken together, Sirt3 deficiency induces a mild, superoxide-dependent endothelial dysfunction in mice fed a high-cholesterol diet. In cultured endothelial cells, a novel C/EBP-β-dependent rescue mechanism maintains net SOD2 activity upon transient knockdown of Sirt3.

No MeSH data available.


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Loss of Sirt3 decreases endothelial SOD2 activity and increases SOD2 expression without affecting other ROS-scavenging or generating systems. a Superoxide dismutase 2 (SOD2) activity based on superoxide dismutating capacity in HAEC following siRNA-mediated knockdown of Sirt3; enzymatic activity was normalized to SOD2 protein expression; medians and single data points are shown. b Overall SOD2 activity, as in a without normalizing to protein content. c SOD2 mRNA (left) and protein (right) expression in HAEC following siRNA-mediated knockdown of Sirt3 using quantitative PCR and western blot, respectively. d Acetylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 using western blot analysis. e Nitrosylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 using western blot analysis. f–h Expression analyses of f catalase, g SOD1, h SOD3, using quantitative PCR; beta actin served as loading control in western blots, representative blots are shown. At least three independent experiments in biological triplicates were performed, scr scrambled control
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Fig3: Loss of Sirt3 decreases endothelial SOD2 activity and increases SOD2 expression without affecting other ROS-scavenging or generating systems. a Superoxide dismutase 2 (SOD2) activity based on superoxide dismutating capacity in HAEC following siRNA-mediated knockdown of Sirt3; enzymatic activity was normalized to SOD2 protein expression; medians and single data points are shown. b Overall SOD2 activity, as in a without normalizing to protein content. c SOD2 mRNA (left) and protein (right) expression in HAEC following siRNA-mediated knockdown of Sirt3 using quantitative PCR and western blot, respectively. d Acetylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 using western blot analysis. e Nitrosylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 using western blot analysis. f–h Expression analyses of f catalase, g SOD1, h SOD3, using quantitative PCR; beta actin served as loading control in western blots, representative blots are shown. At least three independent experiments in biological triplicates were performed, scr scrambled control

Mentions: To unravel the mechanism underlying increased endothelial mitochondrial superoxide levels upon Sirt3 deficiency, we addressed SOD2-specific activity. Following transient knockdown of Sirt3 in HAEC, superoxide scavenging capacity of SOD2 was reduced by threefold compared with controls (Fig. 3a). Unexpectedly, expression levels of SOD2 were increased by 4.4 fold on RNA- and by 2.8 fold on protein level, respectively (Fig. 3c), thus compensating for the Sirt3-dependent loss of SOD2 specific activity; overall endothelial SOD2 activity, without normalizing to protein levels, was unchanged upon transient knockdown of Sirt3 (Fig. 3b). Decreased SOD2-specific activity was associated with a trend towards SOD2 hyperacetylation (Fig. 3d). SOD2 nitrosylation was unchanged upon knockdown of Sirt3 (Fig. 3e), suggesting a Sirt3-dependent, deacetylation-mediated activation of endothelial SOD2 under physiological conditions. Expression levels of other superoxide scavenging or decomposing enzymes, including SOD1, SOD3, catalase, thioredoxin 1, thioredoxin 2, thioredoxin-dependent peroxide reductase (PRDX3), and glutathione peroxidase were unaltered (Fig. 3f–h, Fig S3A–E). Accordingly, the expression level of endothelial superoxide-generating enzyme NADPH oxidase was unaffected by transient knockdown of Sirt3 (Fig S3F, G). Whereas no difference occurred in the cytosolic subunit p47phox (Fig S3F), we observed a slight increase in the mRNA level of the membrane-bound subunit p22phox, which did not translate into an increased protein level (Fig S3G).Fig. 3


Mild endothelial dysfunction in Sirt3 knockout mice fed a high-cholesterol diet: protective role of a novel C/EBP-β-dependent feedback regulation of SOD2.

Winnik S, Gaul DS, Siciliani G, Lohmann C, Pasterk L, Calatayud N, Weber J, Eriksson U, Auwerx J, van Tits LJ, Lüscher TF, Matter CM - Basic Res. Cardiol. (2016)

Loss of Sirt3 decreases endothelial SOD2 activity and increases SOD2 expression without affecting other ROS-scavenging or generating systems. a Superoxide dismutase 2 (SOD2) activity based on superoxide dismutating capacity in HAEC following siRNA-mediated knockdown of Sirt3; enzymatic activity was normalized to SOD2 protein expression; medians and single data points are shown. b Overall SOD2 activity, as in a without normalizing to protein content. c SOD2 mRNA (left) and protein (right) expression in HAEC following siRNA-mediated knockdown of Sirt3 using quantitative PCR and western blot, respectively. d Acetylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 using western blot analysis. e Nitrosylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 using western blot analysis. f–h Expression analyses of f catalase, g SOD1, h SOD3, using quantitative PCR; beta actin served as loading control in western blots, representative blots are shown. At least three independent experiments in biological triplicates were performed, scr scrambled control
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Fig3: Loss of Sirt3 decreases endothelial SOD2 activity and increases SOD2 expression without affecting other ROS-scavenging or generating systems. a Superoxide dismutase 2 (SOD2) activity based on superoxide dismutating capacity in HAEC following siRNA-mediated knockdown of Sirt3; enzymatic activity was normalized to SOD2 protein expression; medians and single data points are shown. b Overall SOD2 activity, as in a without normalizing to protein content. c SOD2 mRNA (left) and protein (right) expression in HAEC following siRNA-mediated knockdown of Sirt3 using quantitative PCR and western blot, respectively. d Acetylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 using western blot analysis. e Nitrosylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 using western blot analysis. f–h Expression analyses of f catalase, g SOD1, h SOD3, using quantitative PCR; beta actin served as loading control in western blots, representative blots are shown. At least three independent experiments in biological triplicates were performed, scr scrambled control
Mentions: To unravel the mechanism underlying increased endothelial mitochondrial superoxide levels upon Sirt3 deficiency, we addressed SOD2-specific activity. Following transient knockdown of Sirt3 in HAEC, superoxide scavenging capacity of SOD2 was reduced by threefold compared with controls (Fig. 3a). Unexpectedly, expression levels of SOD2 were increased by 4.4 fold on RNA- and by 2.8 fold on protein level, respectively (Fig. 3c), thus compensating for the Sirt3-dependent loss of SOD2 specific activity; overall endothelial SOD2 activity, without normalizing to protein levels, was unchanged upon transient knockdown of Sirt3 (Fig. 3b). Decreased SOD2-specific activity was associated with a trend towards SOD2 hyperacetylation (Fig. 3d). SOD2 nitrosylation was unchanged upon knockdown of Sirt3 (Fig. 3e), suggesting a Sirt3-dependent, deacetylation-mediated activation of endothelial SOD2 under physiological conditions. Expression levels of other superoxide scavenging or decomposing enzymes, including SOD1, SOD3, catalase, thioredoxin 1, thioredoxin 2, thioredoxin-dependent peroxide reductase (PRDX3), and glutathione peroxidase were unaltered (Fig. 3f–h, Fig S3A–E). Accordingly, the expression level of endothelial superoxide-generating enzyme NADPH oxidase was unaffected by transient knockdown of Sirt3 (Fig S3F, G). Whereas no difference occurred in the cytosolic subunit p47phox (Fig S3F), we observed a slight increase in the mRNA level of the membrane-bound subunit p22phox, which did not translate into an increased protein level (Fig S3G).Fig. 3

Bottom Line: Sirtuin 3 (Sirt3) is an NAD(+)-dependent mitochondrial deacetylase associated with superoxide dismutase 2 (SOD2)-mediated protection from oxidative stress.Thus, we aimed to unravel the effects of endogenous Sirt3 on endothelial function and oxidative stress.In cultured endothelial cells, a novel C/EBP-β-dependent rescue mechanism maintains net SOD2 activity upon transient knockdown of Sirt3.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, University Heart Center Zurich, University Hospital Zurich, Raemistr. 100, 8091, Zurich, Switzerland. stephan.winnik@usz.ch.

ABSTRACT
Sirtuin 3 (Sirt3) is an NAD(+)-dependent mitochondrial deacetylase associated with superoxide dismutase 2 (SOD2)-mediated protection from oxidative stress. We have reported accelerated weight gain and impaired metabolic flexibility in atherosclerotic Sirt3 (-/-) mice. Oxidative stress is a hallmark of endothelial dysfunction. Yet, the role of Sirt3 in this context remains unknown. Thus, we aimed to unravel the effects of endogenous Sirt3 on endothelial function and oxidative stress. Knockdown of Sirt3 in human aortic endothelial cells (HAEC) increased intracellular mitochondrial superoxide accumulation, as assessed by electron spin resonance spectroscopy and fluorescence imaging. Endothelium-dependent relaxation of aortic rings from Sirt3 (-/-) mice exposed to a normal diet did not differ from wild-type controls. However, following 12 weeks of high-cholesterol diet and increasing oxidative stress, endothelial function of Sirt3 (-/-) mice was mildly impaired compared with wild-type controls. Relaxation was restored upon enhanced superoxide scavenging using pegylated superoxide dismutase. Knockdown of Sirt3 in cultured HAEC diminished SOD2 specific activity, which was compensated for by a CCAAT/enhancer binding protein beta (C/EBP-β)-dependent transcriptional induction of SOD2. Abrogation of this feedback regulation by simultaneous knockdown of C/EBP-β and Sirt3 exacerbated mitochondrial superoxide accumulation and culminated into endothelial cell death upon prolonged culture. Taken together, Sirt3 deficiency induces a mild, superoxide-dependent endothelial dysfunction in mice fed a high-cholesterol diet. In cultured endothelial cells, a novel C/EBP-β-dependent rescue mechanism maintains net SOD2 activity upon transient knockdown of Sirt3.

No MeSH data available.


Related in: MedlinePlus