Limits...
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.


Related in: MedlinePlus

Interruption of the physiological C/EBP-β-dependent transcriptional feedback regulation of SOD2 during transient knockdown of Sirt3 exacerbates mitochondrial superoxide formation and culminates in endothelial cell death. a Fluorescence imaging of HAEC following single and simultaneous transient knockdown of C/EBP-β and Sirt3, respectively. Representative micrographs show nuclei (blue) and mitochondrial superoxide (red, MitoSOX™), the latter visualized by the mitochondrial- and superoxide-specific fluorescent MitoSOX™ probe. Scale bars 20 μm. b Quantification of mitochondrial superoxide per cell; medians and single data points are shown. c, d Representative brightfield phase-contrast micrographs of cultured HAEC 17 h (c) and 40 h (d) after transient knockdown of Sirt3 and C/EBP-β, either alone or in combination; scale bars 200 μm. At least three independent experiments in biological triplicates were performed, scr scrambled control, DAPI 4′-6-diamidin-2-phenylindol, n.s. non-significant, *p < 0.05, **p < 0.01, ***p < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4829622&req=5

Fig6: Interruption of the physiological C/EBP-β-dependent transcriptional feedback regulation of SOD2 during transient knockdown of Sirt3 exacerbates mitochondrial superoxide formation and culminates in endothelial cell death. a Fluorescence imaging of HAEC following single and simultaneous transient knockdown of C/EBP-β and Sirt3, respectively. Representative micrographs show nuclei (blue) and mitochondrial superoxide (red, MitoSOX™), the latter visualized by the mitochondrial- and superoxide-specific fluorescent MitoSOX™ probe. Scale bars 20 μm. b Quantification of mitochondrial superoxide per cell; medians and single data points are shown. c, d Representative brightfield phase-contrast micrographs of cultured HAEC 17 h (c) and 40 h (d) after transient knockdown of Sirt3 and C/EBP-β, either alone or in combination; scale bars 200 μm. At least three independent experiments in biological triplicates were performed, scr scrambled control, DAPI 4′-6-diamidin-2-phenylindol, n.s. non-significant, *p < 0.05, **p < 0.01, ***p < 0.001

Mentions: To reveal the functional relevance of the C/EBP-β-dependent transcriptional feedback regulation of endothelial SOD2 upon Sirt3 deficiency, we assessed mitochondrial superoxide levels following single or simultaneous knockdown of C/EBP-β and Sirt3, respectively, compared with sham-transfected controls. Concomitant with the abrogation of the transcriptional induction of SOD2 following simultaneous knockdown of C/EBP-β and Sirt3, mitochondrial superoxide levels were further enhanced compared with single-knockdown controls (Fig. 6a, b). Transient knockdown of C/EBP-β alone had no effect on mitochondrial superoxide levels (Fig. 6a, b). Interestingly, we observed an increased cell death upon prolonged cultivation (40 h) following simultaneous knockdown of C/EBP-β and Sirt3 that occurred in none of the control conditions (Fig. 6c, d): Incubation for up to 40 h following knockdown led to a demise of the majority of cells (Fig. 6d), which we interpret as the consequence of increased oxidative stress.Fig. 6


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)

Interruption of the physiological C/EBP-β-dependent transcriptional feedback regulation of SOD2 during transient knockdown of Sirt3 exacerbates mitochondrial superoxide formation and culminates in endothelial cell death. a Fluorescence imaging of HAEC following single and simultaneous transient knockdown of C/EBP-β and Sirt3, respectively. Representative micrographs show nuclei (blue) and mitochondrial superoxide (red, MitoSOX™), the latter visualized by the mitochondrial- and superoxide-specific fluorescent MitoSOX™ probe. Scale bars 20 μm. b Quantification of mitochondrial superoxide per cell; medians and single data points are shown. c, d Representative brightfield phase-contrast micrographs of cultured HAEC 17 h (c) and 40 h (d) after transient knockdown of Sirt3 and C/EBP-β, either alone or in combination; scale bars 200 μm. At least three independent experiments in biological triplicates were performed, scr scrambled control, DAPI 4′-6-diamidin-2-phenylindol, n.s. non-significant, *p < 0.05, **p < 0.01, ***p < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829622&req=5

Fig6: Interruption of the physiological C/EBP-β-dependent transcriptional feedback regulation of SOD2 during transient knockdown of Sirt3 exacerbates mitochondrial superoxide formation and culminates in endothelial cell death. a Fluorescence imaging of HAEC following single and simultaneous transient knockdown of C/EBP-β and Sirt3, respectively. Representative micrographs show nuclei (blue) and mitochondrial superoxide (red, MitoSOX™), the latter visualized by the mitochondrial- and superoxide-specific fluorescent MitoSOX™ probe. Scale bars 20 μm. b Quantification of mitochondrial superoxide per cell; medians and single data points are shown. c, d Representative brightfield phase-contrast micrographs of cultured HAEC 17 h (c) and 40 h (d) after transient knockdown of Sirt3 and C/EBP-β, either alone or in combination; scale bars 200 μm. At least three independent experiments in biological triplicates were performed, scr scrambled control, DAPI 4′-6-diamidin-2-phenylindol, n.s. non-significant, *p < 0.05, **p < 0.01, ***p < 0.001
Mentions: To reveal the functional relevance of the C/EBP-β-dependent transcriptional feedback regulation of endothelial SOD2 upon Sirt3 deficiency, we assessed mitochondrial superoxide levels following single or simultaneous knockdown of C/EBP-β and Sirt3, respectively, compared with sham-transfected controls. Concomitant with the abrogation of the transcriptional induction of SOD2 following simultaneous knockdown of C/EBP-β and Sirt3, mitochondrial superoxide levels were further enhanced compared with single-knockdown controls (Fig. 6a, b). Transient knockdown of C/EBP-β alone had no effect on mitochondrial superoxide levels (Fig. 6a, b). Interestingly, we observed an increased cell death upon prolonged cultivation (40 h) following simultaneous knockdown of C/EBP-β and Sirt3 that occurred in none of the control conditions (Fig. 6c, d): Incubation for up to 40 h following knockdown led to a demise of the majority of cells (Fig. 6d), which we interpret as the consequence of increased oxidative stress.Fig. 6

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