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. 2017 Aug 19;6(8):e006114.
doi: 10.1161/JAHA.117.006114.

Sirtuin 3 Deficiency Accelerates Hypertensive Cardiac Remodeling by Impairing Angiogenesis

Tong Wei  1 , Gaojian Huang  2 , Jing Gao  2 , Chenglin Huang  2 , Mengwei Sun  3 , Jian Wu  4 , Juan Bu  5 , Weili Shen  2
Affiliations

Sirtuin 3 Deficiency Accelerates Hypertensive Cardiac Remodeling by Impairing Angiogenesis

Tong Wei et al. J Am Heart Assoc. .

Abstract

Background: Emerging evidence indicates that impaired angiogenesis may contribute to hypertension-induced cardiac remodeling. The nicotinamide adenine dinucleotide-dependent deacetylase Sirtuin 3 (SIRT3) has the potential to modulate angiogenesis, but this has not been confirmed. As such, the aim of this study was to examine the relationship between SIRT3-mediated angiogenesis and cardiac remodeling.

Methods and results: Our experiments were performed on SIRT3 knockout and age-matched wild-type mice infused with angiotensin II (1400 ng/kg per minute) or saline for 14 days. After angiotensin II infusion, SIRT3 knockout mice developed more severe microvascular rarefaction and functional hypoxia in cardiac tissues compared with wild-type mice. These events were concomitant with mitochondrial dysfunction and enhanced collagen I and collagen III expression, leading to cardiac fibrosis. Silencing SIRT3 facilitated angiotensin II-induced aberrant Pink/Parkin acetylation and impaired mitophagy, while excessive mitochondrial reactive oxygen species generation limited angiogenic capacity in primary mouse cardiac microvascular endothelial cells. Moreover, SIRT3 overexpression in cardiac microvascular endothelial cells enhanced Pink/Parkin-mediated mitophagy, attenuated mitochondrial reactive oxygen species generation, and restored vessel sprouting and tube formation. In parallel, endothelial cell-specific SIRT3 transgenic mice showed decreased fibrosis, as well as improved cardiac function and microvascular network, compared with wild-type mice with similar stimuli.

Conclusions: Collectively, these findings suggest that SIRT3 could promote angiogenesis through attenuating mitochondrial dysfunction caused by defective mitophagy.

Keywords: Sirtuin 3; angiogenesis; cardiac remodeling; mitochondria; mitophagy; oxidative stress.

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Figures

Figure 1
Figure 1
Sirtuin 3 (SIRT3) deficiency accelerates angiotensin II (Ang II)–induced cardiac remodeling. A, Heart to body weight (HW/BW ratios, n=10). B, Representative echocardiography images of wild‐type (WT) and SIRT3‐knockout (KO) hearts. C through F, Quantitative analysis of left ventricular end‐systolic diameter (LVESD), left ventricular posterior wall thickness (LVPWs), left ventricular anterior wall thickness (LVAWs), and left ventricular fractional shortening (LVFS) (n=10/group). G, Representative hematoxylin and eosin (H&E), Masson trichrome, Picrosirius red, and collagen I staining of WT and SIRT3‐KO hearts. H, Quantitative analysis of collagen volume fraction. I and J, Representative Western blot and quantitation for (K) α‐smooth muscle actin (α‐SMA) and (L) collagen I and collagen III. Data are presented as mean±SEM. *P<0.05, **P<0.01 vs genotype‐matched sham mice; # P<0.05, ## P<0.01 vs WT‐Ang II mice. Col indicates collagen.
Figure 2
Figure 2
Sirtuin 3 (SIRT3) deficiency exacerbates microvascular rarefaction after angiotensin II (Ang II) challenge. A, Representative micro‐computed tomography images of myocardial vasculatures after Microfil perfusion. B and C, Quantitative assessment of total vessel volume, and intramyocardial vessel volume normalized to total vessel volume (n=3/group). Data are presented as mean±SEM. D, Representative images of cardiac sections stained with lectin (red) and 4’,6‐diamidino‐2‐phenylindole (DAPI; blue). Scale, 200 μm. E, Quantification of lectin intensity. F, Representative images of cardiac sections stained with hypoxyprobe‐1 (green) and DAPI (blue). Scale, 200 μm. G, Quantification of hypoxyprobe‐1 intensity. H through I, Representative vascular endothelial growth factor (VEGF) Western blot and densitometry. Data are relative to that of wild‐type (WT)‐sham mice (mean±SEM). *P<0.05, **P<0.01 vs genotype‐matched sham mice; # P<0.05, ## P<0.01 vs WT‐Ang II mice. KO indicates knockout.
Figure 3
Figure 3
Loss of Sirtuin 3 (SIRT3) aggravates mitochondrial dysfunction and oxidative stress. A and B, Representative SIRT3 Western blot and desnitometry. C, Representative transmission electron microscopy (TEM) images of mitochondria in wild‐type (WT) and SIRT3‐knockout (KO) hearts. D and E, Anti‐4‐hydroxy‐nonenal (4‐HNE), aldehyde dehydrogenase 2 (ALDH2), and manganese superoxide dismutase (MnSOD) Western blot and densitometry. Data are relative to that of WT‐sham control mice (mean±SEM). *P<0.05, **P<0.01 vs genotype‐matched sham mice; # P<0.05 vs WT‐Ang II mice.
Figure 4
Figure 4
Sirtuin 3 (SIRT3) deficiency impairs angiogenic capacity. A, Representative micrographs of microvessel sprouting in aortic rings from wild‐type (WT), SIRT3‐knockout (KO), and endothelium‐specific SIRT3‐transgenic (SIRT3 TgEC) mice. B, Representative tube formation images of cardiac microvascular endothelial cells infected with SIRT3‐short hairpin RNA (shRNA), lentivirus‐mediated SIRT3 (LV‐SIRT3), or negative controls treated with or without angiotensin II (Ang II; 1 μmol/L) for 12 hours. Scale, 500 μm. C, Quantitative analysis of microvessel sprouting in aortic rings. Data are presented as mean±SEM. *P<0.05 vs genotype‐matched sham mice; # P<0.05 vs WT‐Ang II mice. D, Quantitative analysis of tube length in each well. Five images per well were randomly chosen to be measured. Results are expressed as fold‐change over untreated vector‐infected cells. **P<0.01 vs untreated genotype‐matched cells; ## P<0.01 vs Ang II–treated vector‐infected cells.
Figure 5
Figure 5
Loss of Sirtuin 3 (SIRT3) suppresses Pink/Parkin‐mediated mitophagy. A, Representative confocal images of autophagic flux stained with LC3B (red), MitoTracker Green, and 4′,6‐diamidino‐2‐phenylindole (blue). Scale, 200 μm. B, LC3‐II expression was detected by Western blotting with quantitative analysis in SIRT3‐short hairpin RNA (shRNA) or negative control cells. C, LC3‐II expression was detected by Western blotting with quantitative analysis in lentivirus‐mediated SIRT3 (LV‐SIRT3) or negative control infected cells. D, Representative Western blot analysis of Pink1 and parkin following immunoprecipitation with SIRT3 antibody. E, Representative Western blot and (F) quantitative analysis of acetylated Pink1 and acetylated Parkin in SIRT3‐shRNA or negative control cells. G, Representative Western blot and (H) quantitative analysis of acetylated Pink1 and acetylated Parkin in LV‐SIRT3 or negative control cells. Results are expressed as fold‐change over untreated vector‐infected cells. Data are presented as means±SEM. *P<0.05, **P<0.01 vs untreated vector‐infected cells; # P<0.05, ## P<0.01 vs angiotensin II (Ang II)–treated vector‐infected cells. CQ indicates chloroquine.
Figure 6
Figure 6
Attenuated mitochondrial oxidative stress restores angiogenic capacity. A, Representative images of reactive oxygen species (ROS) and MitoTracker Red staining in Sirtuin 3 (SIRT3)‐short hairpin RNA (shRNA) or negative control cardiac microvascular endothelial cells (CMVECs). Scale, 200 μm. B, MitoTracker Red quantification. C, Representative images and (D) quantification of ROS with MitoTracker Red staining in lentivirus‐mediated SIRT3 (LV‐SIRT3) or negative control CMVECs. Scale, 200 μm. E, Representative Western blot analysis and quantification of manganese superoxide dismutase (MnSOD) expression in SIRT3‐shRNA or negative control cells. F, Representative Western blot analysis and (H) quantification of MnSOD expression in LV‐SIRT3 or negative control cells. Results are expressed as fold‐change over untreated vector‐infected cells. *P<0.05, **P<0.01 vs untreated vector‐infected cells; # P<0.05, ## P<0.01 vs angiotensin II (Ang II)–treated vector‐infected cells. G, Representative fluorescence images and (H) quantification of ROS with MitoTracker Red staining in CMVECs pretreated with or without coenzyme Q10. Scale, 200 μm. I, Representative tube formation images of CMVECs pretreated with or without coenzyme Q10. J, Quantitative analysis of tube length in each well. Five images per well were randomly chosen to be measured. *P<0.05, **P<0.01 vs control cells; # P<0.05, ## P<0.01 vs Ang II treatment.
Figure 7
Figure 7
Sirtuin 3 (SIRT3) overexpression reverses hypertensive cardiac remodeling. A, Heart/body weight (HW/BW) ratios in wild‐type (WT) and endothelium‐specific SIRT3‐transgenic (SIRT3‐TgEC) mice (n=10). B, Representative echocardiography images of WT and SIRT3‐TgEC mice hearts. C through F, Quantitative analysis of left ventricular end‐systolic diameter (LVESD), left ventricular posterior wall thickness (LVPW), left ventricular anterior wall thickness (LVAW), and left ventricular fractional shortening (LVFS) (n=10/group). G, Representative hematoxylin and eosin (H&E), wheat germ agglutinin (WGA), Masson trichrome, and Picrosirius red staining of WT and SIRT3‐TgEC mice hearts. H, Quantitative analysis of collagen volume fraction. I, Quantitative analyses of cardiomyocyte cross‐sectional area. J, Representative images of cardiac sections stained with lectin (red) and 4′,6‐diamidino‐2‐phenylindole (DAPI; blue). Scale, 200 μm. K, Quantification of lectin intensity. L, Representative images of cardiac sections stained with hypoxyprobe‐1 (green) and DAPI (blue). Scale, 200 μm. M, Quantification of hypoxyprobe‐1 intensity. Data are presented as mean±SEM. *P<0.05, **P<0.01 vs genotype‐matched sham mice; # P<0.05, ## P<0.01 vs WT‐angiotensin II (Ang II) mice.
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