Sirt3 Impairment and SOD2 Hyperacetylation in Vascular Oxidative Stress and HypertensionNovelty and Significance

Measurements of mitochondrial O₂^· – in aorta (A), endothelial NO (B), SOD2 acetylation (C), and densitometry ratio of Acetyl-SOD2 to total SOD2 (D) in Sirt3^−/− and wild-type (WT) mice. After 14 days of saline or low dose of angiotensin II (Ang II; 0.3 mg/kg per day) infusion, mice were euthanized for aorta isolation for O₂^· – measurements by MitoSOX and high-performance liquid chromatography (HPLC; A), NO analysis by electron spin resonance (ESR; B),⁵ or Western blot analysis (C). Figure shows representative Western blots. *P<0.05 vs WT, **P<0.01 vs WT+Ang II (n=6).

Angiotensin II (Ang II)-induced hypertension (A) and aortic mitochondrial O₂^· – (B) in wild-type (WT) and mCAT mice. After 14 days of saline or Ang II (0.7 mg/kg per day) infusion, mice were euthanized for isolation of aorta for O₂^· – measurements by MitoSOX and high-performance liquid chromatography (HPLC). *P<0.01 vs sham, **P<0.01 vs Ang II (n=8). ESR indicates electron spin resonance.

SOD2 acetylation (A), aortic SOD2 activity (B), and Sirt3 S-glutathionylation in wild-type (WT) and mCAT mice. After 14 days of saline or angiotensin II (Ang II; 0.7 mg/kd per day) infusion, mice were euthanized for isolation of aorta and kidney. SOD2 acetylation and SOD2 activity were measured in the homogenate of aortic vessel by Western blot and electron spin resonance, correspondingly. Sirt3 S-glutathionylation was determined in isolated kidney mitochondria by Sirt3 immunoprecipitation and Western blot with GSH (glutathione, reduced form) antibodies. Figure shows representative blots from 3 experiments. *P<0.01 vs sham, **P<0.01 vs Ang II (n=8).

SOD2 acetylation (A), Sirt3 expression, and mitochondrial O₂^· – (B through D) in human aortic endothelial cells treated with angiotensin II (Ang II; 10 nmol/L), TNFα (1 nmol/L), or Ang II+TNFα for 24 h prior analysis. Mitochondrial O₂^· – was measured by mitochondria-targeted probe MitoSOX and accumulation of O₂^· –-specific product 2-OH-Mito-E⁺ using high-performance liquid chromatography (HPLC; B, insert).^5,51 Human aortic endothelial cells (HAEC) were transfected with scramble or Sirt3 siRNA and were stimulated with Ang II+TNFα before Western blot analysis and measurements of mitochondrial O₂^· –.⁵ Ang II and TNFα cooperatively induce SOD2 acetylation and stimulate production of mitochondrial O₂^· –, which is attenuated by scavenging of mitochondrial H₂O₂. *P<0.01 vs control, **P<0.01 vs Ang II+TNFα.

Inhibition of angiotensin II (Ang II)-induced hypertension and vascular dysfunction by mitochondria-targeted antioxidants. A, Systolic blood pressure in mice infused with saline (sham), Ang II (0.7 mg/kg per day) or treated with mitoEbselen after onset of Ang II-induced hypertension, (B) Western blot of SOD2 acetylation, (C) systolic blood pressure, and (D) acetylcholine-dependent relaxation of aortic vessels isolated from mice infused with Ang II and treated with mitoTEMPO (1.4 mg/kg per day) after onset of hypertension. Results represent mean±SEM (n=4–8). *P<0.01 vs Ang II, **P<0.05 vs wild type (WT)+Ang II (n=4–8).

Comparison of aortic SOD2 acetylation (A), aortic SOD2 activity (B), and angiotensin II (Ang II)-induced hypertension in wild-type (WT), mCAT, or Sirt3^−/− mice (C). After 14 days of saline or Ang II (0.7 mg/kg per day) infusion, mice were euthanized for isolation of aorta for analysis for Western blot and electron spin resonance analysis of SOD2 acetylation and SOD2 activity⁵ in aortic homogenates. Equal protein loading in acetyl-SOD2 study was confirmed by β-actin Western blot. Results are mean±SEM (n=4–6). *P<0.01 vs WT (sham), **P<0.01 vs WT+Ang II, ^#P<0.05 vs WT+Ang II.